Biological materials and methods useful in the diagnosis and treatment of diseases

ABSTRACT

The present invention relates to a method of making a β-form of a prion protein which preferably has more β-sheet than α-helix structure and is soluble in the absence of a denaturant and/or is non-aggregated and exhibits partial resistance to digestion with proteinase K. The invention also relates to use of the β-form in medicine, especially for raising antibodies useful in the treatment and/or diagnosis of prion diseases. The invention also relates to methods of screening for compounds which are capable of inhibiting and/or reversing the conversion of the native α-form of a prion protein to a β-form, and to uses of identified compounds in medicine.

The present invention relates to prion proteins.

Prions are infectious pathogens that differ from bacteria, fungi,parasites, viroids, and viruses, both with respect to their structureand with respect to the diseases that they cause. Molecular biologicaland structural studies of prions promise to open new vistas intofundamental mechanisms of cellular regulation and homeostasis notpreviously appreciated. Kuru, Creutzfeldt-Jakob disease (CJD), fatalfamilial insomnia (FFI) and Gerstmann-Sträussler-Scheinker syndrome(GSS) are all human neurodegenerative diseases that are caused by prionsand are frequently transmissible to laboratory animals. Familial CJD andGSS are also genetic disorders. No effective therapy exists to preventthese fatal disorders².

In addition to the prion diseases of humans, disorders of animals areincluded in the group of known prion diseases. Scrapie of sheep andgoats is the most studied of the prion diseases. Bovine spongiformencephalopathy (BSE) is thought to result from abnormal feedingpractices. BSE threatens the beef industry of Great Britain and possiblyother countries; the production of pharmaceuticals involving cattle isalso of concern. Control of sheep scrapie in many countries is apersistent and vexing problem².

Since 1986, more than 170,000 cattle have developed BSE in GreatBritain. Many investigators contend that BSE, often referred to as “madcow disease”, resulted from the feeding of dietary protein supplementsderived from rendered sheep offal infected with scrapie to cattle, apractice banned since 1988. It is thought that BSE will disappear withthe cessation of feeding rendered meat and bone meal, as has been thecase in kuru of humans, confined to the Fore region of New Guinea andonce the most common cause of death among women and children. Kuru hasalmost disappeared with the cessation of ritualistic cannibalism.

Prion diseases are associated with the accumulation of a conformationalisomer (PrP^(Sc)) of host-derived prion protein (PrP^(c)) with anincrease in its β-sheet content¹. According to the protein-onlyhypothesis, PrP^(Sc) is the principal or sole component of transmissibleprions². Although the structure of PrP^(c) has been determined³ and hasbeen found to consist predominantly of α-helices, the insolubility ofPrP^(Sc), which is isolated from tissue in a highly aggregated state andwhich has a high β-sheet content, has precluded high-resolutionstructural analysis. Various workers have attempted to make forms of PrPwhich are intermediate between the normal (PrP^(c)) form and theabnormal, pathogenic form (PrP^(Sc)), having a predominantly β-sheetform therefore termed the β-form.

Hornemann & Glockshuber PNAS 95, 6010-6014 (1998)⁸ describe aβ-intermediate which is an unfolding intermediate of mouse PrP andcontains predominantly β-sheet elements of secondary structure asopposed to α-helix. Swietnicki et al (1997) J. Biol. Chem. 272:44,October 31 pp27517-27520 describe an identical folding intermediatederived from human PrP⁹⁰⁻²³¹. The mouse β-intermediate is derived fromoxidised PrP which contains the native disulphide bond. The mouse PrPintermediate required urea (a denaturant) for stabilisation. Thereference on page 6011 “Results” states that the mouse β-intermediateexhibits stability at pH 4.0 in the absence of denaturant; however thisis based upon an equilibrium calculation. The free energy of folding(Table 1, page 6012) is approximated from a fit of the equationdescribed in Materials and Methods (page 6011) to the data in FIG. 1A.From this an equilibrium constant can be calculated which describes thesmall proportion of molecules that will exist as the β-intermediate inthe absence of denaturant. The proportion of molecules in this state islow (around 0.2%) and nothing can be said about their solubility in theabsence of denaturant as they are not detectable. Indeed one would arguethey are extremely unlikely to be soluble in the absence of denaturantbecause folding intermediates are structural states that are populatedduring rearrangement of a polypeptide chain from a random structure to adefined native conformation, or vice versa. They are characterised ashaving native-like secondary structure, few tertiary interactions,increased molecular volume, increased side chain mobility and exposedhydrophobic residues. These properties combined make them prone toaggregation and, as such, are generally insoluble in the absence ofdenaturants. Several references describe these properties indetail¹⁸⁻²³.

Moreover, the above calculation is dependent upon the transition being agenuine equilibrium, ie. fully reversible. If the transition is notreversible this analysis is invalid. We have performed similarexperiments and have found that full reversibility is abolished atprotein concentrations in excess of 1 mg/ml, with refolding yields<100%.

Zhang et al (1997) Biochem 36:12, 3543-3553 describe a β-sheet form ofrecombinant Syrian hamster PrP containing residues 90-231 which isformed by a method involving refolding at a pH of 6.5. It is clear frompage 3548, second column and FIG. 7, that the β-form described isneither monomeric nor soluble in aqueous solution.

According to a first aspect the invention provides a method of making aβ-form of a prion protein which has more β-sheet than α-helix structure,can exist as a monomer and can retain solubility in aqueous solution inthe s absence of a denaturant, the method comprising:

providing a reduced prion protein which does not include a disulphidebond and causing the conformation of the protein to change so that itadopts the β-form.

Preferably, the change in conformation is caused by exposure toconditions of acidic pH, preferably a pH of 5.5 or less, more preferablya pH of 4.8 or less and most preferably a pH of 4.0.

Skilled persons will appreciate that the β-sheet and α-helix structurecan be shown by circular dichroism spectropolarimetry as describedherein. While the native prion protein state is characterised by astrong α-helical signal, the β-form of the invention shows a shift to aconformation dominated by β-sheet. By “dominated” in this context weinclude the meaning that there is more β-sheet structure of the prionprotein than α-helix structure.

By “exist as a monomer” we include the meaning that the β-form of theprion protein does not exist as an aggregate of two or more β-form prionproteins. Skilled persons will appreciate that analytical sedimentationstudies can be used to determine whether or not a protein exists insolution as a monomer or as an aggregate of two or more proteins. Asuitable technique is described in Zhang et al (1997) Biochem, 36:12,3542-3553 (see page 3545-3546 passage entitled AnalyticalSedimentation). The technique involves the use of an analyticalultracentrifuge (Beckman Optimat XL-A) equipped with a six channel cell,using ultraviolet absorption between 220 and 280 nm.

By “can retain solubility in the absence of a denaturant” we include themeaning that a significant proportion eg around 30% or more of theβ-form remains in solution as a monomer after centrifugation at 100,000g for 1 hour and preferably 150,000 g for 8-16 hours, most preferably at200,000 g for 8-16 hours. The centrifugation may be carried out on a 2mg/ml aqueous solution of the β-form prion protein comprising NaAcetate+10 mM Tris. HCl+pH 4.0 at 25° C. The structural characteristicsof the remaining protein in solution can be determined by circulardichroism spectropolarimetry, for example.

Preferably, the β-form remains soluble without denaturant to aconcentration of more than 1 mg/ml, more preferably at least 12 mg/ml,and especially more than 20 mg/ml.

It will of course be appreciated that the above requirement for theβ-form to be capable of retaining solubility in the absence of thedenaturant in no way limits the invention to methods or compositionswhich do not include a denaturant.

A β-form of a prion protein of the invention also comprises a prionprotein which has at least 20% of its residues in β-sheet structure,more preferably at least 50% and most preferably 50 to 60% or more, asdetermined by CD spectropolarimetry.

A β-form of a prion protein of the invention also comprises a prionprotein which is non-aggregated and exhibits partial resistance toproteinase K digestion.

A β-form of a prion protein of the invention also comprises a prionprotein which is non-aggregated but is capable of forming an aggregatedfibrous and/or amyloid form, preferably on exposure to a denaturant.

Preferably, a β-form of a prion protein of the invention also comprisesa prion protein which is non-aggregated but is capable of forming anon-fibrillar aggregate on exposure to conditions of sufficient ionicstrength. Preferably, the non-fibrillar aggregate is capable of forminga fibrillar structure.

By “conditions of sufficient ionic strength” we mean an ionic strengthcapable of converting the non-aggregated β-form to an aggregated form.For example, salt concentrations of 50 mM to 500 mM, especially 100 mMor more are sufficient to cause murine β-form prion protein to form anon-fibrillar aggregate. A particularly preferred salt concentration is100-200, more preferably 150 mM eg NaCl or KCl.

A β-form of a prion protein of the invention also comprises a prionprotein which is capable of interconverting between a β-form as definedherein and an α-form of a prion protein as described herein.

A β-form of a prion protein of the invention may exhibit one or more ofthe above properties.

In another aspect, the invention provides a method of obtainingnon-aggregated β-form from a sample comprising partially digesting thesample with proteinase K.

It will be appreciated that by “prion protein” is included variants,fragments and fusions that have interactions or activities which aresubstantially the same as those of a full length prion protein sequence,but which may be more convenient to use, for example in an assay. A“variant” will have a region which has at least 70% (preferably 80, 90,95 or 99%) sequence identity with the 91-231 region of native human PrPsequence described herein or the corresponding region in the PrP ofother species as measured by the Bestfit Program of theWisconsin-Sequence Analysis Package, version 8 for Unix. The percentageidentity may be calculated by reference to a region of at least 50 aminoacids (preferably at least 75, 100, 120 or 140) of the candidate variantmolecule, and the most similar region of equivalent length in the native91-231 region, allowing gaps of up to 5%.

The percent identity may be determined, for example, by comparingsequence information using the GAP computer program, version 6.0described by Devereux et al. (Nucl. Acids res. 12:387, 1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Neddleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math 2.482. 1981). The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Bribskov and Burgess, Nucl. Acids Res.14:6745, 1986 as described by Schwarts and Dayhoff, eds, Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

Hybrid prion proteins comprising amino acid sequences from two or moredifferent species also fall within the scope of the term “prion protein”used herein. Hybrid proteins comprising protein domains from differentspecies can be produced using known recombinant DNA techniques, such asthose described in WO93/20093 in relation to hybrid human/porcine factorVIII proteins.

A “fragment” comprises at least 50, preferably 75, 100, 120 or 130 aminoacids of the native 91-231 sequence.

Such activities will include the abilities mentioned herein, such as theability to be soluble without denaturant and may include the ability toraise antibodies and for use in screening compounds in accordance withthe following aspects of the invention and the ability to form anaggregated fibrous and/or amyloid form especially a non-fibrillaraggregate which preferably comprises spherical particles having adiameter of from approx 10-20 nM which can be visualised by electronmicroscopy, when exposed to suitable conditions etc.

Preferably, the β-form of a prion protein exhibits partial resistance todigestion with proteinase K(PK).

By “partial resistance to digestion with proteinase K (PK)” we includethe meaning that after incubation of 1 mg/ml of the protein in 10 mMNaAcetate+10 mM Tris. Acetate, pH 8.0 with 0.5 μg/ml PK (based on thetotal digestion reaction volume) at 37° C. for 30 mins some protein canbe shown to be undigested when subjected to SDS-PAGE as describedherein. Preferably, the majority of the protein is undigested.

Preferably, the β-form of the invention displays resistance to digestionat increased concentrations of PK eg 5 μg/ml PK or more.

The disease-related isoform of PrP, PrP^(Sc), is distinguishedbiochemically from the normal cellular isoform of the protein, PrP^(c),by its partial resistence to digestion with the enzyme proteinase K. Wehave now demonstrated that not only aggregated β-PrP is proteaseresistant but also that the soluble β-PrP monomer is also PK-resistantand to a level approximating to that seen with PrP^(Sc). This is strongevidence to support the contention that β-PrP may be the precursor ofPrP^(Sc).

The novel β-form, or an aggregate of two or more β-forms, of theinvention may be used to prepare antibodies which selectively recognisethe β-form (whether aggregated or not) rather than the α-form or viceversa.

By “α-form” of a prion protein we include the meaning of a prion proteinwhich has more α-helical than β-sheet structure. The α-form may also becharacterised by sensitivity to degradation by proteinase K.

Any reductant and conditions which allow reduction can be used in themethod of the invention as long as they do not cause irreversiblemodification to the polypeptide chain. Reduction of a disulphide bondcan be determined by Ellman's assay (Ellman, G. L., 1959, Arch Biochem &Biophys). Reduction of the disulphide bond preferably takes place beforethe pH is lowered. The acidic pH at which conformation change takesplace may be approximately pH 5.5 or less, and preferably pH 4.8 orless, most preferably a pH of 4.0. Skilled persons will appreciate thatany buffer that is effective around pH 4.0 can be used, such as 10 mMNaAcetate+10 mM Tris.Acetate.

Preferably, the β-form has substantially the same molecular volume(measured by size exclusion chromatography) as the native form of theprion protein.

In a second aspect, the invention provides a preparation of a β-form ofa prion protein wherein at least 1% of the β-form can exist as a monomerand can retain solubility in aqueous solution in the absence of adenaturant. Preferably, the β-form is obtainable by a method accordingto the first aspect of the invention.

The invention also provides the above (soluble, undenatured) β-form of aprion protein for use in medicine, preferably in the prevention,treatment and/or diagnosis of a prion disease.

It will be appreciated that by virtue of properties such as itssolubility, the β-form is amenable to high resolution structuralanalysis and so has particular utility for research into the mechanismsof prion disease especially prion replication. Such utility is not foundin known insoluble forms of prion proteins.

The prion disease may be selected from one or more of the diseasesaffecting humans. Alternatively or additionally, the prion diseases areselected from one or more of the diseases which affect domestic farmanimals such as cows, sheep and goats. Other prion diseases includetransmissible mink encephalopathy; chronic wasting disease of mule deerand elk, bovine spongiform encephalopathy and, more recently, a wholeseries of new animal diseases that are thought to have arisen from theirdietary exposure to the BSE agent. These include feline spongiformencephalopathy, affecting domestic cats and captive wild-cats (such ascheetahs, pumas, ocelots, tigers) and spongiform encephalopathies ofcaptive exotic ungulates (including kudu, nyala, gemsbok, eland).

Preferably, the prion protein is selected from human, bovine or ovineprion proteins, more preferably human prion protein.

According to a third aspect of the invention there is provided a methodof making an antibody against a prion protein having a β-form as definedin accordance with the earlier aspects of the invention, comprisingadministering said β-form to an animal and collecting and purifying thedirectly or indirectly resulting antibody. The antibody may bepolyclonal, but is preferably monoclonal.

By “antibody” in accordance with the invention we include moleculeswhich comprise or consists of antigen binding fragments of an antibodyincluding Fab, Fv, ScFv and dAb. We also include agents whichincorporate such fragments as portions for targetting prion moleculesand/or cells or viruses which display such molecules.

According to this aspect of the invention, there is also provided amonoclonal antibody capable of distinguishing between the native α-formand the β-form of a prion protein as defined in accordance with earlieraspects of the invention or vice versa. Also provided is a hybridomacell capable of producing such a monoclonal antibody.

In accordance with this aspect of the invention there is also providedan antibody for use in medicine, which antibody binds preferentially tothe β-form of a prion protein rather than to the α-form of the prionprotein or vice versa. Preferably, the antibody is for use in themanufacture of a composition for use in the prevention, treatment and/ordiagnosis of a prion disease.

According to a fourth aspect of the invention there is provided a methodof detecting the presence of a prion protein having a β-form as definedin accordance with the earlier aspects of the invention in a biologicalsample. The method preferably comprises providing an antibodypreparation comprising an antibody which preferentially binds the β-formrather than the α-form and detecting whether the antibody binds β-form.

Conveniently, the antibody is directly or indirectly labelled bysuitable means and its binding to the β-form is detected by detecting alabel.

Preferably, the biological sample comprises or consists of a bodilyfluid or tissue such as blood or blood derivative, ie a component suchas plasma, lymphoid tissue (such as tonsils, appendices, lymph orspleen), cerebrospinal fluid faeces, urine, lymph or sputum. Thebiological sample may be a tissue sample eg a biopsy tissue sample.

It may be advantageous to introduce an anti-β-form antibody into one ofthe tissues mentioned above either to detect β-form or to remove β-formbefore it reaches the brain. Such anti-β-form antibodies are preferablyantibodies which preferentially react with the β-form rather than thenormal α-form of the prion protein.

By “preferentially” according to the various aspects of the invention weinclude the meaning that the ratio of α/β binding may be 45/55, 25/75,more preferably, 10/90, 5/95, 1/99 or substantially 0/100.

The invention also provides a method of detecting antibodies in abiological sample, which antibodies bind preferentially to a β-form of aprion protein rather than the α-form comprising exposing the β-form tothe biological sample to permit binding of antibody to the β-form anddetecting the binding of antibody to the β-form. Optionally, the β-formis immobilised before exposure to the sample.

The invention also provides a method of obtaining a β-form binding agentwhich binds preferentially to a β-form of a prion protein rather than anα-form comprising exposing the β-form to a sample to permit binding ofagents to the β-form and optionally collecting the agent bound to theβ-form. Optionally, the β-form is immobilised before exposure to thesample. Preferably, the binding agent is directly or indirectly labelledand its binding to the β-form is detected by detecting the label.

The invention also provides a kit useful for diagnosing a prion diseasefrom a biological sample comprising a binding agent, preferably anantibody, which is capable of preferentially binding the β-form ratherthan the α-form, or a β-form of a prion protein which binds said bindingagent; and means for detecting binding of the binding agent to theβ-form. The binding agent or β-form being coupled optionally to an inertsupport. Preferably, the means for detecting binding comprises aradioactive, enzymic or fluorescent label.

The invention also provides an in vitro method for diagnosing apredisposition to, or the presence of, a prion disease comprisingproviding a reduced α-form of a prion protein, preferably at a pH ofaround 5.5 or less, preferably pH 4.8 or less, most preferably a pH of4.0; comparing the amount or rate of formation of a β-form as definedherein in the presence and absence of a biological sample eg from apatient. Increased rate or amount of β-form formation is indicative of apredisposition to, or the presence of, a prion disease.

The invention also provides a method of treating a biological sample toremove a β-form of a prion protein comprising providing a binding agentwhich binds preferentially to the β-form of a prion protein rather thanto the α-form of the prion protein, exposing the biological sample tothe binding agent whereby a β-form of a prion protein can bind thebinding agent and optionally collecting the treated biological sample.Preferably, the binding agent is immobilised before the exposure to thesample.

The invention also provides a method of diagnosing a predisposition to,or the presence of, a prion disease comprising providing a β-form of aprion protein; providing a biological sample; and exposing the solutionto the sample and detecting the presence of an aggregation of theβ-form, such an aggregation being indicative of predisposition to, orthe presence of, a prion disease.

Preferably, the aggregation of the β-form is a non-fibrillar aggregatewhich preferably comprises spherical or irregularly shaped particleshaving a diameter of from 10-20 nm which can be visualised by electronmicroscopy.

The invention also provides the use of a β-form or a non-fibrillaraggregate thereof in the manufacture of a composition for use as avaccine against a prion disease. A vaccine composition of the inventionpreferably comprises a β-form or a non-fibrillar aggregate thereof andan adjuvant.

According to a fifth aspect of the invention there is provided a methodof identifying an agent that is capable of preventing, reducing and/orreversing the conversion of a prion protein to a β-form as definedabove, the method comprising: providing a sample of a prion protein andcomparing the amount of the β-form quantitatively or qualitatively inthe presence and absence of a test agent.

In a sixth aspect of the invention, there is provided a method ofidentifying an agent that is capable of preventing or reducing theconversion of a prion protein from the β-form, as defined in accordancewith earlier aspects of the invention, to an aggregated fibrous and/oramyloid form, especially a non-fibrillar aggregate mentioned above, themethod comprising providing a solution containing the β-form andcomparing qualitatively or quantitatively the amount of the aggregatedand/or amyloid form produced in the presence and absence of a testagent.

Preferably, the amount of the aggregated and/or amyloid, especiallynon-fibrillar aggregate, form is measured using a spectrofluorimeter.

In a seventh aspect of the invention there is provided an agent which isidentifiable by a method as defined in accordance with the fifth orsixth aspect of the invention.

In an eighth aspect the invention provides an agent capable ofpreventing, reducing and/or reversing the conversion of a prion proteinfrom an α-form to a β-form as defined in accordance with earlier aspectsof the invention.

In a ninth aspect the invention provides an agent capable of preventingor reducing the conversion of a β-form of a prion protein as defined inaccordance with earlier aspects of the invention to an aggregated and/oramyloid , especially non-fibrillar aggregate, form.

The agents according to the seventh, eighth and ninth aspects of theinvention may be a drug-like compound or lead compound for thedevelopment of a drug-like compound. Thus, the methods may be methodsfor identifying a drug-like compound or lead compound for thedevelopment of a drug-like compound that is capable of preventing,reducing and/or reversing the conversion of a prion protein to a β-form;and/or that is capable of preventing and/or reducing the conversion ofthe β-form to an aggregated and/or amyloid, especially non-fibrillaraggregate, form.

The term “drug-like compound” is well known to those skilled in the art,and may include the meaning of a compound that has characteristics thatmay make it suitable for use in medicine, for example as the activeingredient in a medicament. Thus, for example, a drug-like compound maybe a molecule that may be synthesised by the techniques of organicchemistry, less preferably by techniques of molecular biology orbiochemistry, and is preferably a small molecule, which may be of lessthan 5000 daltons molecular weight and which may be water-soluble. Adrug-like compound may additionally exhibit features of selectiveinteraction with a particular protein or proteins and be bioavailableand/or able to penetrate target cellular membranes, but it will beappreciated that these features are not essential.

The term “lead compound” is similarly well known to those skilled in theart, and may include the meaning that the compound, whilst not itselfsuitable for use as a drug (for example because it is only weakly potentagainst its intended target, non-selective in its action, unstable,poorly soluble, difficult to synthesise, too toxic or has poorbioavailability) may provide a starting-point for the design of othercompounds that may have more desirable characteristics.

The compounds identified in the methods of the invention may themselvesbe useful as a drug or they may represent lead compounds for the designand synthesis of more efficacious compounds.

In another aspect the invention provides an agent that comprises abinding agent portion which binds preferentially to the β-form of theprion protein rather than the α-form, and an effector portion which iscapable of one or more of the following functions: (1) preventing,reducing and/or reversing the conversion of a prion protein to a β-form;(2) preventing or reducing the conversion of a prion protein from theβ-form to an aggregated fibrous and/or amyloid, especially anon-fibrillar aggregate form; or (3) destroying a β-form of a prionprotein and/or a cell or virus displaying such a protein.

Preferably, the binding agent portion comprise an antibody or a fragmentthereof. Preferably the antibody or fragment thereof is made accordingto aspects of the present invention.

In one preferred embodiment the effector portion of an agent comprises acompound of the earlier aspects of the invention.

In another preferred embodiment the agent comprises an effector portionwhich is directly or indirectly cytotoxic.

By a “directly cytotoxic” portion we include a portion of an agent whichis in itself toxic to the cell if it reaches, and preferably enters, thesaid cell.

By an “indirectly cytotoxic” portion we include a portion of an agentwhich can be converted into or produce a cytotoxic agent by the actionof a further reagent, or which can convert a substantially non-toxicsubstance into a toxic substance. We also include a portion of an agentwhich can bind specifically to a compound which is directly orindirectly cytotoxic.

Non-limiting examples of cytotoxic portions include a drug, pro-drug,radionuclide, protein including an enzyme, antibody or any othertherapeutically useful reagent, including cytokines such as tumournecrosis factor, interleukin-2 or interferon-γ.

Thus, the drug may be a cytotoxic chemical compound such asmethotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), daunorubicin or other intercalating agents. The protein maybe ricin. The cytotoxic portion may comprise a highly radioactive atom,such iodine-131, rhenium-186, rhenium-188 or yttrium-90.

The enzyme, or enzymatic portion thereof, may be directly cytotoxic,such as DNaseI or RNase, or indirectly cytotoxic such as an enzyme whichconverts a substantially non-toxic pro-drug into a toxic form. Theenzyme cytosine deaminase converts 5-fluorocytosine (5FC) to5-fluorouracil (5FU) (Mullen et al (1922) PNAS 89, 33); the herpessimplex enzyme thymidine kinase sensitises cells to treatment with theantiviral agent ganciclovir (GCV) or aciclovir (Moolten (1986) CancerRes. 46, 5276; Ezzedine et al (1991) New Biol 3, 608). The cytosinedeaminase of any organism, for example E. coli or Saccharomycescerevisiae, may be used. Examples of the construction of antibody-enzymefusions are disclosed by Neuberger et al (1984) Nature 312, 604.

Other examples of pro-drug/enzyme combinations include those disclosedby Bagshawe et al (WO 88/07378), namely various alkylating agents andthe Pseudomonas spp. CPG2 enzyme, and those disclosed by Epenetos &Rowlinson-Busza (WO 91/11201), namely cyanogenic pro-drugs (for exampleamygdalin) and plant-derived α-glucosidases. The nitroreductase/CB1954system described by Bridgewater et al (1995) Eur. J. Cancer 31A,2362-2370 is another example of an enzyme/prodrug combination suitablefor use in the invention.

In a tenth aspect the invention provides an agent in accordance with theearlier aspects of the invention for use in medicine. Preferably, use ofthe aspects in the manufacture of a composition for use in theprevention, treatment and/or diagnosis of a prion disease, or for use asa research reagent.

In an eleventh aspect the invention provides a pharmaceuticalcomposition comprising a pharmaceutically effective amount of an agentin accordance with the seventh, eighth and/or ninth aspects of theinvention, together with a pharmaceutically acceptable diluent orcarrier.

In a twelfth aspect the invention provides a method of preventing and/ortreating a prion disease comprising administering to a subject aneffective amount of an agent in accordance with the earlier aspects ofthe invention. By “effective amount” we include the meaning thatsufficient quantities of the agent are provided to produce a desiredpharmaceutical effect beneficial to the health of the recipient.

For a better understanding, the following non-limiting examples whichembody certain aspects of the invention will now be described withreference to the following figures.

FIG. 1

(a) Secondary and tertiary structure of the two human PrP isoforms. Themain graph shows CD spectra collected in the far UV region.

Oxidised human PrP at pH 8.0 is shown in open circles and displays atypically α-helical spectrum with 47% of amide residues involved inhelical structure¹⁷. In contrast reduced human PrP at pH 4.0 displays aβ-sheet spectrum, shown in open triangles. There is little or no helixpresent with up to 40% of amide residues adopting a β-sheetconformation¹⁸. The inset displays near UV CD spectra for oxidised humanPrP pH 8.0 (open circles), reduced human PrP pH 4.0 (open triangles) anddenatured human PrP (open squares). The oxidised protein clearlydisplays a high level of tertiary organisation in the aromatic region ofthe spectrum, whereas the denatured PrP lacks any distinct tertiaryinteractions. The reduced human PrP displays a level of tertiaryorganisation intermediate between native and denatured states.

(b) ¹H NMR spectra of the upfield regions of the α- and β-forms ofhuPrP⁹¹⁻²³¹. Peaks upfield of 0.7 ppm are characteristic of strongtertiary interactions between methyl groups and aromatic rings found infolded, globular proteins.

(c) Expanded region of a ¹H, ¹⁵N HSQC spectrum of the β-form ofhuPrP⁹¹⁻²³¹ showing its chemical shift dispersion, which is much reducedrelative to the α-form (Hornemann S. and Glockshuber R., J Mol Biol,261, 614-619 (1996)).

While the ID ¹H-NMR spectrum of native human PrP⁹¹⁻²³¹ exhibits widechemical shift dispersion characteristic of a fully folded globularprotein, the 1D ¹H and ¹H ¹⁵N HSQC spectra of the β-form of PrP exhibitconsiderably less chemical shift dispersion (FIG. 1b,c). This lack ofdispersion is characteristic of the loss of fixed side chaininteractions, which, in conjunction with the aromatic CD results,suggests some similarities with molten globule states. In addition,proton and nitrogen line-widths of the β-form (FIG. 1c) are comparableto those observed in the folded and unfolded regions of the α-PrPconformation indicating that the β-form is monomeric at the extremelyhigh concentrations required for NMR, thus confirming the gel-filtrationresults. The mobile unstructured regions of β-PrP have been assignedfrom the sharpness and height of the peaks. We find that residues 91-126and 229-230 are mobile in β-PrP, moreover, this is the same region thatis unstructured in the α-PrP conformation. Hence, the rearrangement fromα-helix to β-sheet must occur within the structured region of thecellular conformation.

FIG. 2

Determination of the apparent molecular weight of PrP by size exclusionchromatography.

(a) Elution profile of molecular weight standards used to construct acalibration curve of molecular weight versus elution time (not shown).(b) Oxidised human PrP pH 8.0 in the alpha form elutes with an apparentmolecular weight of 18 kDa. This excess weight (calculated mass is 16248kDa) is due to the large molecular volume of PrP resulting from thedispersed secondary structure elements. (c) Reduced human PrP pH 4.0 inthe β-form also elutes as a monomer with an apparent molecular weight of18 kDa. (d) Oxidised human PrP at pH 4.0 partially denatured with 1MGuHCl. Addition of 1M GuHCl to oxidised human PrP at pH 4.0 results inaggregation and precipitation. Clarified supernatant contains adenatured form of PrP with an increased molecular volume correspondingto an apparent molecular weight of 40 kDa.

FIG. 3

β-PrP is more prone to form high molecular weight aggregates than α-PrP.Right angle light scattering of a 1 mg/ml solution of α-PrP (opencircles) shows there are no high molecular weight aggregates formed uponaddition of GuHCl. In contrast β-PrP, which is highly soluble in aqueousbuffer alone, readily forms high molecular weight aggregates upon theaddition of low concentration of GuHCl (open triangles). Maximumprecipitation occurs at 0.4 M GuHCl, with subsequent re-dissolution ofaggregates at higher concentrations of denaturant.

FIG. 4

β-PrP aggregates self-assemble into fibrils. The protein aggregatesappear in two forms by negative stain electron microscopy. (A) The mostcommon form is small (about 10 nm diameter) irregularly shaped and isseen in all samples. (B) The other aggregation form is fibrils which areincreasingly prevalent the longer the sample is incubated. These fibrescan be seen to intertwine, again a phenomenon that increases with time.Scale bars shown in white represent a length of 200 nm. In order tocomply with safety regulations governing the handling of prion protein,electron microscopy was performed on mouse PrP⁹¹⁻²³¹ treated in anidentical manner to the human protein.

β-PrP, at a concentration of 0.27 mg/ml in 20 mM sodium acetate pH4, wastreated with 1/9 volumes of a 5M stock of GuHCl to give a final proteinand denaturant concentrations of 0.25 mg/ml and 0.5 M respectively. Theprocedure for staining the protein is as follows. A dilute solution ofPrP (^(˜)2 μl) is dropped onto the grid and the molecules adhere to thecarbon film. Bonding to the surface prevents interactions betweenprotein molecules. The sample is then flooded with 2% uranyl acetate w/vwhich coats the carbon surface and any particles stuck to it. The excessis blotted off leaving a thin film. This procedure seldom, if ever,leads to aggregation owing to the initial adherence to the grid surface.In our hands, when doing extensive single molecule work, we have notseen aggregation phenomena using this method. Further, when the PrPmolecule is initially laid down the particles are small and circular andonly produce fibrils after several hours. If the laying down processcaused the aggregation we would not see this time-dependent behaviour.

FIG. 5

β-Prp displays partial PK resistance in monomeric and aggregated states.α-PrP is sensitive to PK digestion and is completely digested at 0.5μg/ml PK. The concentrations of PK indicated are the finalconcentrations in the digestion reactions.

Using identical conditions for digestion in which β-PrP remains solubleand monomeric (data not shown), soluble β-PrP has partial resistance toproteinase K with the majority of protein undigested at 0.5 μg/ml.Aggregated β-PrP possesses increased resistance to PK digestion withsome protein surviving intact at 5 μg/ml PK. The concentrations of PKindicated are the final concentrations in the digestion reactions.Although β-PrP reverts to α-PrP at pH8.0 this process requires severaldays for completion. Within the timescale of PK digestion the proteinremains as β-PrP.

FIG. 6

Known prion protein sequences from other mammalian species, using thesingle letter code for amino acids as follows:

A=Ala; D=Asp; E=Glu, F=Phe; K=Lys; L=Leu; M=Met; N=Asn; P=Pro; Q=Gly;R=Arg; S=Ser; T=Thr; and V=Val.

Such information is available from databases such as EMBL, Genbank,Swis-Prot, Brookhaven.

METHODS 1. Purification of Human PrP

Plasmid Design and Protein Expression

The open reading frame of the human PrP gene was amplified by PCR usingoligonucleotide primers designed to create an unique N-terminal BamHIsite and C-terminal HindIII site for directional cloning of the fragmentinto the expression vector pTrcHisB (Invitrogen Corp.). The primercorresponding to the N-terminal region of PRNP to be expressed wasdesigned to mutate a glycine at codon 90 to methionine, with theC-terminal primer replacing a methionine residue at 232 to a stop codon.

Human PrP Open Reading Frame: SEQ ID NO. 35

1 ATGGCGAACC TTGGCTGCTG GATGCTGGTT CTCTTTGTGG CCACATGGAG 51 TGACCTGGGCCTCTGCAAGA AGCGCCCGAA GCCTGGAGGA TGGAACACTG 101 GGGGCAGCCG ATACCCGGGGCAGGGCAGCC CTGGAGGCAA CCGCTACCCA 151 CCTCAGGGCG GTGGTGGCTG GGGGCAGCCTCATGGTGGTG GCTGGGGGCA 201 GCCTCATGGT GGTGGCTGGG GGCAGCCCCA TGGTGGTGGCTGGGGACAGC 251 CTCATGGTGG TGGCTGGGGT CAAGGAGGTG GCACCCACAG TCAGTGGAAC301 AAGCCGAGTA AGCCAAAAAC CAACATGAAG CACATGGCTG GTGCTGCAGC 351AGCTGGGGCA GTGGTGGGGG GCCTTGGCGG CTACATGCTG GGAAGTGCCA 401 TGAGCAGGCCCATCATACAT TTCGGCAGTG ACTATGAGGA CCGTTACTAT 451 CGTGAAAACA TGCACCGTTACCCCAACCAA GTGTACTACA GGCCCATGGA 501 TGAGTACAGC AACCAGAACA ACTTTGTGCACGACTGCGTC AATATCACAA 551 TCAAGCAGCA CACGGTCACC ACAACCACCA AGGGGGAGAACTTCACCGAG 601 ACCGACGTTA AGATGATGGA GCGCGTGGTT GAGCAGATGT GTATCACCCA651 GTACGAGAGG GAATCTCAGG CCTATTACCA GAGAGGATCG AGCATGGTCC 701TCTTCTCCTC TCCACCTGTG ATCCTCCTGA TCTCTTTCCT CATCTTCCTG 751 ATAGTGGGAT GA

N-terminal Sense Oligo: SEQ ID NO. 36

5′-TTTG GAT CCG ATG CAA GGA GGT GGC ACC CAC-3′

C-terminal Antisense Obligo: SEQ ID NO. 37

5′-CAA GAA GCT TTC AGC TCG ATC CTC TCT GG-3′

The ligated pTrcHisB/PRNP construct was used to transform the E. colihost strain BL21 (DE3) (Novagen), genotype F′ ompT hsdS_(B) (r_(B)⁻m_(B) ⁻) gal dcm (DE3) which was then plated onto Luria-Bertoni (LB)agar plates containing 100 μg/ml carbenicillin. Following growthovernight at 37° C. single colonies were picked and used to inoculate10×10 ml of LB broth containing 100 μg/ml carbenicillin. This culturewas grown overnight at 37° C. with vigorous shaking. The 10 ml cultureswere used as inocula for 10×1 litre of LB broth containing 100 μg/mlcarbenicillin which had been pre-warmed to 37° C. Growth at 37° C. withvigorous shaking was allowed to progress until the culture reached anOD₆₀₀ of 0.6. Expression was then induced by addition ofisopropyl-β-D-galactopyranoside to a final concentration of 1 mM and theculture resupplemented with carbenicillin to a level of 100 μg/ml.Following 4 hours of induced growth the cells were harvested bycentrifugation at 8,500 rpm for 10 minutes.

Extraction, Refolding and Purification of Recombinant Human PrP

The cell pellet was resuspended in 50 ml of lysis buffer (50 mM Tris. ClpH 8.0, 200 mM NaCl, 0.1% Triton X100, 10 μg/ml DNase 1, 10 μg/mllysozyme) and disrupted by sonication in 1 minute bursts for a total of5 minutes. Centrifugation at 9,600 rpm for 30 minutes pelleted all theinsoluble material and the supernatant was discarded. The pellet wasthen washed twice by resuspension in 50 ml of lysis buffer withcentrifugation at 7,500 rpm for 5 minutes between each wash.Solubilisation of protein in the pellet was performed by resuspension in50 ml of 50 mM Tris. Cl, 6M GuHCl, 100 mM DTT pH 8.0. Cell debris andinsoluble material was removed by centrifugation at 9,600 rpm for 30minutes. The supernatant was clarified by passage through a 0.2 μmfilter and loaded onto a 20 ml Ni-NTA-Sepharose (Quiagen) columnpre-equilibrated with 50 mM Tris. Cl, 6M GuHCl pH 8.0.

After washing the column with the above buffer, bound protein was elutedwith a 15 column volume linear gradient of 0 mM to 300 mM imidazole inloading buffer. Recombinant PrP eluted at 185 mM imidazole. Elutedfractions were pooled and oxidation of disulphides was achieved byvigorous stirring in the presence of 1 μM CuSO₄ and dissolvedatmospheric oxygen for 16 hours. PrP containing oxidised disulphides wasseparated from reduced protein using reverse phase chromatography on anRP304-C4 column. The protein was loaded in 50 mM Tris.Cl, 6M GuHCl pH8.0, washed with ddH₂O+0.1% trifluoroacetic acid (TFA) and eluted with alinear gradient of 15% to 60% acetonitrile+0.09% TFA. Human PrP emergedas two major peaks; oxidised protein at 40% acetonitrile and a secondpeak containing reduced PrP eluted at 45% acetonitrile. The oxidisedpeak fractions were pooled and neturalised by the addition of 1M Tris.ClpH 8.0 to a final concentration of 100 mM and saturated ammoniumsulphate added to a final concentration of 70%. Precipitated PrPaccumulated at the interface between organic and aqueous phases and wasremoved to a separate container. The protein was solubilised in aminimal volume of 50 mM Tris.Cl, 6M GuHCl pH 8.0 and then dilutedrapidly to a protein concentration of 1 mg/ml and dialysed for 16 hoursagainst 50 mM Tris.Cl pH 8.0 with a buffer change after 8 hours.Following dialysis the N-terminal fusion peptide was removed by additionof enterokinase at lunit/3 mg protein. Cleavage was allowed to occur at37° C. for 14 hours and terminated by the addition of “proteasecomplete” (Boehringer Mannheim Corp).

Final purification was carried out by applying the protein material to a10 ml S-Sepharose FastFlow column equilibrated with 25 mM Tris.Cl pH 7.0and following a 5 column volume wash with the same buffer, protein waseluted with a 10 column volume linear gradient of 0 mM to 300 mM NaCl.Recombinant PrP lacking the N-terminal fusion peptide eluted at 150 mMwhilst uncleaved material remained bound until 250 mM NaCl. Elutedfractions were concentrated in an Amicon cell with a 10 kDa cut offmembrane and then dialysed overnight against 25 mM Tris.Cl pH 7.0, 0.02%NaAzide containing a small amount of activated charcoal. Sucrose wasadded to 5% w/v and the protein snap frozen in liquid nitrogen for longterm storage at −80° C.

Recombinant human PrP in the oxidised α-form was purified as describedabove and dialysed into 10 mM NaAcetate+10 mM Tris.HCl pH 8.0. Toconvert this material to the β-form the protein was reduced anddenatured in 100 mM DTT in 6M GuHCl+10 mM NaAcetate+10 mM Tris.HCl pH8.0 for 16 hrs. The protein was refolded by dialysis against 10 mMNaAcetate+10 mM Tris.HCl+1 mM DTT pH4.0 and precipitated materialremoved by centrifugation at 150,000 g for 8 hrs. Protein concentrationwas determined by UV absorption using a calculated molar extinctioncoefficient of 19632 M⁻¹ cm⁻¹ at 280 nm.

2. Determination of Aggregation State of PrP by Gel Filtration

A Bio-Sil 125-5 size exclusion column (BioRad) was equilibrated with theappropriate buffer at a flow rate of 1 ml/min producing a back pressureof 900 psi. A 20 μl (360 μg) aliquot of molecular weight standards(BioRad) containing markers of 670 kDa, 158 kDa, 44 kDa, 17 kDa and 1.35kDa was loaded onto the column equilibrated with 10 mM NaAcetate+10 mMTris.HCl+50 mM NaCl. The markers were eluted with 2 column volumes (30ml) of the same buffer and used to construct a calibration curve for thecolumn. The α-PrP was loaded in a volume of 100 μl (200 μg) and elutedwith 30 mls of 10 mM NaAcetate+10 mM Tris.HCl+50 mM NaCl pH 8.0. β-PrPwas loaded in volume of 100 μl (200 μg) and eluted with 30 mls of NaAcetate+100 mM Tris.HCl+50 mM NaCl pH 4.0.

3. Circular Dichroism Spectropolarimetry

For circular dichroism (CD) measurements 62.5 μM protein was incubatedat 10 mM NaAcetate+10 mM Tris.HCl at either pH 8.0 (α-Prp) or pH 4.0(β-PrP) and molecular ellipticity ([θ], degree M⁻¹ cm⁻¹ was recorded inthe far UV range between 190 nm and 250 nm, using a xenon light sourcein a Jobin-Yvon CD6 spectrometer (cell path length 0.01 cm, slit width1.0 nm; 2 nm bandwidth, integration time 20 sec). Near UV CD spectrawere recorded between 250 nm and 310 nm using 62.5 μM protein in a 10 nmpathlength cuvette with a slit width of 1.0 nm (2 nm bandwidth,integration time 20 sec). All data were recorded at 25° C.

4. NMR Spectroscopy

NMR spectra. shown were acquired at 293 K on a Bruker DRX-500spectrometer. Sample conditions were as follows, α-form: 1 mM humanPrP⁹¹⁻²³¹ in 20 mM sodium acetate-d₃, 2 mM sodium azide, (10% D₂O (v/v))pH 5.55; β-form: 0.75 mM human PrP⁹¹⁻²³¹ in 20 mM sodium acetate-d₃, 2mM sodium azide, (10% D₂O (v/v)) pH 4. 1D ¹H NMR spectra were acquiredwith an acquisition time of 656 ms; ¹H, ¹⁵N HSQC spectra withacquisition times of 328 ms and 168 ms in the direct and indirectdimentions respectively. NMR data were processed using Felix 97(Molecular Simulations Inc). Proton chemical shifts were referencedindirectly to TSP via the water signal.

5. Aggregation of 62 -PrP Observed by Right Angle Light Scattering

Either oxidised human PrP pH 8.0 was diluted to 1 mg/ml in 2 mls of 10mM NaAcetate+10 mM Tris.HCl pH 8.0, or reduced human PrP pH 4.0 wasdiluted to 1 mg/ml in 2 mls of the same buffer at pH 4.0. The presenceof aggregated material was monitored by right angle light scattering ina Schimadzu RF-5301 PC spectrofluorimeter with both excitation andemission monochromators set to slit width of 3 nm. 30 μl aliquots of 6MGuHCl were added and the solution allowed to equilibrate for a fewminutes before each reading was taken. All data were collected at 25° C.

6. Electron Microscopy

Reduced protein refolded at pH 4.0 to form β-sheet structure wasexamined using electron microscopy (EM). The specimens were preparedusing standard negative stain procedures. Three microlitres of proteinsolution at a concentration of 0.25 mg/ml were pipetted onto carbonfilms mounted on copper EM grids. After one minute the grids were washedwith 80 microlitres of aqueous 2% uranyl acetate. The stain was left forapproximately 10 sec before being blotted with filter paper. The gridswere then inserted into a JEOL 1200 transmission electron microscope.Electron micrographs at approximately 1 micron underfocus were recordedon Kodak SO-163 film under normal exposure conditions at 40,000×magnification (calibrated against a grating) at 120 KeV. The defocus ofthe negatives was confirmed by optical diffractometry.

7. Digestion with Proteinase K

Both α-PrP and β-PrP as a monomer and aggregate were subjected todigestion with varying concentrations of proteinase K (BDH) at 37° C.for 1 hr. Protein was digested at a concentration of 1 m/ml in 10 mMNaAcetate+10 mM Tris. Acetate pH 8.0. Digestion was terminated by theaddition of Pefablock (Boehringer Mannheim Corp.) to a finalconcentration of 1 mM. Following the adition of Pefabloc samples wereheated to 100° C. for 5 mins in the presence of SDS loading buffer.Aliquots of 20 μl were subjected to SDS-PAGE and the gels stained withCoomassie brilliant blue.

Here we demonstrate the reversible interconversion of recombinant humanPrP between the native α-form, characteristic of PrP^(c), and asimilarly compact, highly soluble, monomeric form rich in β-structurewhich is stable in aqueous solution. Such an interconversion of aprotein chain between two, discrete, monomeric backbone topologies isunprecedented. We further show that this soluble β-form (β-PrP) is adirect precursor of fibrillar structures that are closely similar tothose isolated from diseased brains. The conversion of PrP^(c) to β-PRPin suitable cellular compartments, and its subsequent stabilisation byintermolecular associated, provides a possible molecular mechanism forprion propagation.

Human PrP⁹¹⁻²³¹ was expressed to high levels in E. coli as a proteinaggregate and solubilised by extraction with 6 M guanidinium chlorideand reducing agent. Subsequent purification, removal of denaturant andoxidation yielded a highly soluble, monomeric protein with a singleintact disulphide bridge. Analysis of this refolded material by circulardichroism (CD) spectropolarimetry revealed a structure rich in aα-helical content (47%) with little β-sheet (18%) (FIG. 1a legend).One-dimensional ¹H nuclear magnetic resonance (NMR) spectra (FIG. 1b)and two-dimensional ¹H-¹⁵N correlation NMR spectra (data not shown) ofthis material show it to be conformationally similar to the previouslydetermined mouse and hamster prion proteins^(3,4), and a previouslycharacterised human Prp⁹¹⁻²³¹ construct⁵.

In common with mouse PrP⁶, human PrP⁹¹⁻²³¹ folds and unfolds through afreely reversible transition (ΔG=−5.6 Kcal./mol) between the fullynative state and a random coil, with no detectable equilibriumintermediates. However, reduction of the disulphide bond in humanPrp⁹¹⁻²³¹, and lowering the pH to 4.0 in a dilute acetate buffer in theabsence of additives, generates a highly soluble protein which can beconcentrated to at least 12 mg/ml. When the reduced protein is subjectedto gel filtration, it elutes as a monomeric species (FIG. 2). The CDsignal in the amide region of the spectrum (FIG. 1a) shows that thishighly soluble reduced species adopts a radically different conformationfrom PrP^(c). While the native state is characterised by a strongα-helical signal, the reduced form shows the shift to a conformationdominated by β-sheet. This constitutes the first observation of asoluble monomeric β-form of the prion protein which opens up theopportunity for biophysical study.

This type of secondary structural transition has been well-documented inproteins that undergo a switch from a soluble monomeric state to anaggregated fibrous and/or amyloid form in which β-structure isstabilised by inter-molecular interactions⁷. However, it isunprecedented for a protein to undergo such a β-sheet conversion whileremaining in a monomeric state at high protein concentrations and in theabsence of denaturants. This is in contrast to the β-intermediate ofmouse PrP¹²¹⁻¹²³ ⁸ which required the presence of denaturant forstabilisation. A similar folding intermediate of human α-PrP⁹¹⁻²³¹exists but is poorly soluble. Clarified material has an increasedapparent molecular weight of 40 kDa (FIG. 2), indicative of tertiarydisorder and expanded molecular volume. Using the amide CD signal alone,it is uncertain whether the non-native compact conformation of humanβ-PrP⁹¹⁻²³¹ is sufficiently condensed to have immobilised side-chainscharacteristic of the native state of orthodox, globular proteins.However, the aromatic region of CD spectra contains signals fromaromatic side-chains in asymmetric environments. Compared to the native,oxidised molecule, the β-form retains a signal from aromatic residuesbut the intensity is diminished (FIG. 1a). This result indicates thatpacked tertiary interactions present in PrP^(c) have been weakened, butnot lost, in the β-conformation. Similarly, gel filtration of thereduced state reveals that is has, within the resolution of thetechnique, the same level of compactness as the PrP^(c) conformation(FIG. 2).

From the above measurement it is not clear whether the reduced form ofthe protein is classifiable as a molten globule or whether it is betterdescribed as an alternative, fully folded conformation with well-definedtertiary interactions between side-chains. The term ‘molten globule’ wasfirst used to describe distinct states adopted by some protein moleculeswhen exposed to mildly denaturing conditions such as moderateconcentrations of chaotropic agents (urea or guanidinium chloride) oracidic pH⁹. The chief signatures of the molten globule state are a wellorganised pattern of native-like backbone (secondary) structure withdisordered side-chains and poorly defined tertiary interactions¹⁰.Originally, they were defined as equilibrium states but as moreinformation became available on the behaviour of transiently populated,kinetic intermediates in folding reactions, often referred to as‘I-states’ the definition has become blurred. This uncertainty isexplained by the fact that I-states and molten globules have the abovefeatures in common, except that the former, kinetic intermediates arepopulated in native conditions. Despite this distinction, it has beenshown for a number of proteins that molten globule states and I-statesare experimentally indistinguishable¹¹. Moreover, because the I-statecan be considered to be the denatured conformation in physiologicalconditions, it has attracted much attention with the context of cellularprocesses such as chaperone-assisted folding, protein transport betweencellular compartments and amyloidosis.

Due to exposure of normally buried non-polar residues, it is rare fornon-native states to show high solubility in the absence of denaturants.However, the availability of the β-form of PrP as a monomeric species ata concentration of 0.75 mM provided the opportunity of examining itsphysical properties using NMR. While the 1D ¹H-NMR spectrum of nativehuman PrP⁹¹⁻²³¹ exhibits wide chemical shift dispersion characteristicof a fully folded globular protein, the spectrum of the β-form of PrPexhibits considerably less chemical shift dispersion. This lack ofdispersion is characteristic of the loss of fixed side chaininteractions, a defining feature of molten globule states¹²⁻¹⁴. However,residual dispersion appears to be greater than that expected for a fullyunfolded protein (FIG. 1b), implying some degree of tertiary packing inthe β-form. This finding is consistent with the reduced but significantCD signal for the β-form in the aromatic region of the spectrum (FIG.1a). Therefore coupled with the amide CD data (FIG. 1b), the NMRchemical shift data points to the β-form being predominantly moltenglobular in nature. In addition, proton line-widths of the β-form arecomparable to those observed in the native PrP^(c) conformationindicating that it is monomeric at the extremely high concentrationsrequired for NMR and confirming the gel-filtration results.

The switch from α-to-β conformation is reversible. When the reducedβ-form is exposed to a higher pH (8.0), the native α-conformation isrestored. However, the rates of inter-conversion, in either direction,are extremely slow, requiring a period of days for completion (data notshown). This high kinetic barrier, however, can be side-stepped by fullydenaturing and refolding at the appropriate pH to generate eitherisoform.

By “fully denaturing” we include the meaning that there is no detectablesecondary or tertiary structure ie the protein forms a “random coil”.Such denaturation can be determined by Circular Dichroism and/or NMRspectroscopy as described herein and can be achieved, for example, bymaintaining the prion protein in 100 mM DTT in 6M GuHCl+10 mMNaAcetate+10 mM NaAcetate+10 mM Tris. HCl pH 8.0 for 16 hours.

Solubility of the two isoforms is not equivalent. The α-form of PrP canbe titrated with the denaturant guanidine hydrochloride (GuHCl) in orderto determine equilibrium parameters for the folding pathway (data notshown). However, while the β-form of PrP is also highly soluble inaqueous buffers, titration with GuHCl leads to inter-molecularassociations resulting in a visible precipitate (FIG. 3). This material,when examined at high magnification, is initially composed of irregularspherical particles (FIG. 4a) which associate over several hours to formfibrils (FIG. 4b), very similar in appearance to those identified indiseased tissue.

PrP^(Sc) is characterised by its partial resistance to digestion withproteinase K (PK). As with native PrP^(c), α-PrP is extremely sensitiveto digestion with PK (FIG. 5). However, β-PrP shows marked proteaseresistance. This PK resistance is a function of the structuralre-organisation of the monomeric β-form, with only a moderate furtherincrease associated with aggregation (FIG. 5). The different patterns ofproteolytic cleavage fragments seen on PK digestion of α-PrP and β-PrPprovide further evidence of a major conformational re-arrangement inβ-PrP. In marked contrast, the partially structured β-sheet conformationof reduced hamster PrP⁹⁰⁻²³¹ reported by Mehlhorn at al¹⁸ and Zhang etal (1997) Biochem, 36:12, 3542-3553¹⁹ is fully sensitive to PKdigestion.

Unusually for a protein with a predominantly helical fold, the majorityof residues in PrP⁹¹⁻²³¹ have a preference for β-conformation (55% ofnon-glycine/proline residues). In view of this property, it is possiblethat the PrP molecule is delicately balanced between radically differentfolds with a high energy barrier between them; one dictated by localstructural propensity (the β-conformation) and one requiring the precisedocking of side-chains (the native α-conformation). Such a balance wouldbe influenced by mutations causing inherited human prion diseases¹⁵. Itis also worthy of note that individuals homozygous for valine atpolymorphic 129 of human PrP (where either methionine or valine can beencoded) are more susceptible to iatrogenic CJD¹⁶, and valine has a muchhigher β-propensity than does methionine. Our results lend support tosuch a hypothesis by showing that the molecule is capable of slowinter-conversion between a native a and a non-native β conformation.Furthermore, we demonstrate that the β-form can be locked byintermolecular association, thus supplying a plausible mechanism ofpropagation of a rare conformational state. It is possible that thePrP^(c) to β-PrP conversion we describe here, caused by reduction andmild acidification, is relevant to the conditions that PrP^(c) wouldencounter within the cell, following its internalisation duringre-cycling. Such a mechanism could underlie prion propagation, andaccount for the transmitted, sporadic and inherited aetiologies of priondisease. Initiation of a pathogenic self-propagating conversionreaction, with accumulation of aggregated β-PrP, may be induced byexposure to a ‘seed’ of aggregated β-PrP following prion inoculation, oras a rare stochastic conformational change, or as an inevitableconsequence of expression of a pathogenic PrP^(c) mutant which ispredisposed to form β-PrP.

8. Antibody Production Method

Methods for purification of antigens and antibodies are described inScopes, R. K. (1993) Protein purification 3rd Edition.Publisher—Springer Verlag. ISBN 0-387-94072-3 and 3-540-94072-3. Thedisclosure of that reference, especially chapters 7 and 9, isincorporated herein by reference.

Antibodies may be produced in a number of ways.

1 The aberrant form of the prion protein eg β-form or aggregatedthereof, especially a non-fibrillar aggregate, is purified from the samespecies as the immunization animal but will usually be human. Theaberrant form may alternatively be prepared by purifying (from theanimal or from a transferred host cell) the non-aberrant form andconverting it to the aberrant form. The immunisation animal may be a“knock-out” mouse, with no prion protein at all. For monoclonalantibodies the animal is normally a mouse; for polyclonal, a rabbit orgoat.

2. Raise antibodies to the antigen. For polyclonal antibodies, this issimply a matter of injecting suitably prepared sample into the animal atintervals, and testing its serum for the presence of antibodies (fordetails, see Dunbar, B. S. & Schwoebel, E. D. (1990) Preparation ofpolyclonal antibodies. Methods Enzymol. 182, 663-670). But it isessential that the antigen (ie. the protein of interest) be as pure aspossible. For monoclonal antibodies, the purity of the antigen isrelatively unimportant if the screening procedure to detect suitableclones uses a bioassay.

Antibodies can also be produced by molecular biology techniques, withexpression in bacterial or other heterologous host cells (Chiswell, D.J. & McCafferty, J. (1992) Phage antibodies: will new “coli-clonal”antibodies replace monoclonal antibodies?” Trends Biotechnol. 10,80-84). The purification method to be adopted will depend on the sourcematerial (serum, cell culture, bacterial expression culture, etc.) andthe purpose of the purification (research, diagnostic investigation,commercial production).

The major methods are as follows:

1. Ammonium sulphate precipitation. The γ-globulins precipitate at alower concentration than most other proteins, and a concentration of 33%saturation is sufficient. Either dissolve in 200 g ammonium sulphate perlitre of serum, or add 0.5 vol of saturated ammonium sulphate. Stir for30 minutes, then collect the γ-globulin fraction by centrifugation,redissolve in an appropriate buffer, and remove excess ammonium sulphateby dialysis or gel filtration.

2. Polyethylene glycol precipitation. The low solubility of γ-globulinscan also be exploited using PEG. Add 0.1 vol of a 50% solution of PEG6,000 to the serum, stir for 30 minutes and collect the y-globulins bycentrifugation. Redissolve the precipitate in an appropriate buffer, andremove excess PEG by gel filtration on a column that fractionates in arange with a minimum around 6,000 Da.

3. Isoelectric precipitation. This is particularly suited for IgMmolecules, and the precise conditions will depend on the exactproperties of the antibody being produced.

4. Ion-exchange chromatography. Whereas most serum proteins have lowisoelectric points, γ-globulins are isoelectric around neutrality,depending on the exact properties of the antibody being produced.Adsorption to cation exchangers in a buffer of around pH 6 has been usedsuccessfully, with elution with a salt gradient, or even standard salinesolution to allow immediate therapeutic use.

5. Hydrophobic chromatography. The low solubility of γ-globulinsreflects their relatively hydrophobic character. In the presence ofsodium or ammonium sulphate, they bind to many hydrophobic adsorbents,such as “T-gel” which consists of β-mercaptoethanol coupled to divinylsulphone-activated agarose.

6. Affinity adsorbents. Staphylococcus aureus Outer coat protein, knownas Protein A, is isolated from the bacterial cells, and it interactsvery specifically and strongly with the invariant region (F_(c)) ofimmunoglobulins (Kessler, S. W. (1975) Rapid isolation of antigens fromcells with a staphylococcal protein A-antibody absorbent: Parameters ofthe interaction of antibody-antigen complexes with protein A. J Immunol.115, 1617-1624. Protein A has been cloned, and is available in manydifferent forms, but the most useful is as an affinity column: Protein Acoupled to agarose. A mixture containing immunoglobulins is passedthrough the column, and only the immunoglobulins adsorb. Elution iscarried out by lowering the pH; different types of IgG elute atdifferent pHs, and so some trials will be needed each time. Thedifferences in the immunoglobulins in this case are not due so much tothe antibody specificity, but due to different types of F_(c) region.Each animal species produces several forms of heavy chain varying in theF_(c) region; for instance, mouse immunoglobulins include subclassesIgG₁, IgG_(2a), and IgG₃ all of which behave differently on elution fromProtein A. Some γ-globulins do not bind well to Protein A. Analternative, Protein G from G from a Streptococcus sp., can be used.This is more satisfactory with immunoglobulins from farm animals such assheep, goats and cattle, as well as with certain subclasses of mouse andrabbit IgGs.

The most specific affinity adsorbent is the antigen itself. The processof purifying an antibody on an antigen adsorbent is essentially the sameas purifying the antigen on an antibody adsorbent. The antigen iscoupled to the activated matrix, and the antibody-containing sampleapplied. Elution requires a process for weakening the antibody-antigencomplex. This is particularly useful for purifying a specific antibodyfrom a polyclonal mixture.

Monoclonal antibodies (MAbs) can be prepared to most antigens. Theantigen-binding portion may be a part of an antibody (for example a Fabfragment) or a synthetic antibody fragment (for example a single chainFv fragment [ScFv]). Suitable monoclonal antibodies to selected antigensmay be prepared by known techniques, for example those disclosed in“Monoclonal Antibodies: A manual of techniques”, H Zola (CRC Press,1988) and in “Monoclonal Hybridoma Antibodies: Techniques andApplications”, J G R Hurrell (CRC Press, 1982). {PRIVATE}

Chimaeric antibodies are discussed by Neuberger et al (1988, 8thInternational Biotechnology Symposium Part 2, 792-799).

Suitably prepared non-human antibodies can be “humanized” in known ways,for example by inserting the CDR regions of mouse antibodies into theframework of human antibodies. The variable heavy (V_(H)) and variablelight (V_(L)) domains of the antibody are involved in antigenrecognition, a fact first recognised by early protease digestionexperiments. Further confirmation was found by “humanisation” of rodentantibodies. Variable domains of rodent origin may be fused to constantdomains of human origin such that the resultant antibody retains theantigenic specificity of the rodent parental antibody (Morrison et al(1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).

That antigenic specificity is conferred by variable domains and isindependent of the constant domains is known from experiments involvingthe bacterial expression of antibody fragments, all containing one ormore variable domains. These molecules include Fab-like molecules(Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al(1988) Science 240, 1038); single-chain Fv (ScFv) molecules where theV_(H) and V_(L) partner domains are linked via a flexible oligopeptide(Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl.Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprisingisolated V domains (Ward et al (1989) Nature 341, 544). A general reviewof the techniques involved in the synthesis of antibody fragments whichretain their specific binding sites is to be found in Winter & Milstein(1991) Nature 349, 293-299.

By “ScFv molecules” we mean molecules wherein the V_(H) and V_(L)partner domains are linked via a flexible oligopeptide.

The advantages of using antibody fragments, rather than wholeantibodies, are several-fold. The smaller size of the fragments may leadto improved pharmacological properties, such as better penetration ofsolid tissue. Effector functions of whole antibodies, such as complementbinding, are removed. Fab, Fv, ScFv and dAb antibody fragments can allbe expressed in and secreted from E. coli, thus allowing the facileproduction of large amounts of the said fragments.

Whole antibodies, and F(ab′)₂ fragments are “bivalent”. By “bivalent” wemean that the said antibodies and F(ab′)₂ fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv and dAb fragments aremonovalent, having only one antigen combining sites.

A CDR-grafted antibody may be produced having at least one chain whereinthe framework regions are predominantly derived from a first antibody(acceptor) and at least one CDR is derived from a second antibody(donor), the CDR-grafted antibody being capable of binding to the β-formPrP antigen. {PRIVATE}

The CDR-grafted chain may have two or all three CDRs derived from thedonor antibody.

Advantageously, in the CDR-grafted chain, the or each CDR comprises acomposite CDR comprising all the residues from the CDR and all theresidues in the corresponding hypervariable region of the donorantibody.

Preferably, at least one residue in the framework regions of theCDR-grafted chain has been altered so that it corresponds to theequivalent residue in the antibody, and the framework regions of theCDR-grafted chain are derived from a human antibody.

Advantageously, the framework regions of the CDR-grafted chain arederived from a human Ig heavy chain. For such heavy chains, it ispreferred that residue 35 in the heavy chain framework regions bealtered so that it corresponds to the equivalent residue in the donorantibody.

Suitably, for such heavy chains, at least one composite CDR comprisingresidues 26 to 35, 50 to 65 or 95 to 102 respectively is grafted ontothe human framework. It will be appreciated in this case that residue 35will already correspond to the equivalent residue in the donor antibody.

Preferably, residues 23, 24 and 49 in such heavy chains correspond tothe equivalent residues in the antibody. It is more preferred thatresidues 6, 23, 24, 48 and 49 in such heavy chains correspond to thedonor antibody in equivalent residue positions. If desired, residues 71,73 and 79 can also so correspond.

To further optimise affinity, any one or any combination of residues 57,58, 60, 88 and 91 may correspond to the equivalent residue in the donorantibody.

The heavy chain may be derived from the human KOL heavy chain. However,it may also be derived from the human NEWM or EU heavy chain.

Alternatively, the framework regions of the CDR-grafted chain may bederived from a human kappa or lambda light chain. For such a lightchain, advantageously at least one composite CDR comprising residues 24to 34, 50 to 56 or 89 to 97 respectively is grafted onto the humanframework. Preferably, residue 49 also corresponds to the equivalentresidue in the donor antibody.

To further optimise affinity, it is preferable to ensure that residues49 and 89 correspond to the equivalent residues in the donor antibody.It may also be desirable to select equivalent donor residues that formsalt bridges.

The light chain is preferably derived from the human REI light chain.However, it may also be derived from the human EU light chain.

Preferably, the CDR-grafted antibody comprises a light chain and a heavychain, one or, preferably, both of which have been CDR-grafted inaccordance with the principles set out above for the individual lightand heavy chains.

It is advantageous that all three CDRs on the heavy chain are alteredand that minimal alteration is made to the light chain. It may bepossible to alter none, one or two of the light chain CDRs and stillretain binding affinity at a reasonable level.

It will be appreciated that in some cases, for both heavy and lightchains, the donor and acceptor residues may be identical at a particularposition and thus no change of acceptor framework residue will berequired.

It will also be appreciated that in order to retain as far as possiblethe human nature of the CDR-grafted antibody, as few residue changes aspossible should be made. It is envisaged that in many cases, it will notbe necessary to change more than the CDRs and a small number offramework residues. Only in exceptional cases will it be necessary tochange a larger number of framework residues.

Preferably, the CDR-grafted antibody is a complete Ig, for example ofisotype IgG₁, or IgG₂, IgG₃ or IgM.

If desired, one or more residues in the constant domains of the Ig maybe altered in order to alter the effector functions of the constantdomains.

Preferably, the CDR-grafted antibody has an affinity for the β-form PrPantigen of between about 10⁵.M⁻¹ to about 10¹².M⁻¹, more preferably atleast 10⁸.M⁻¹.

Advantageously, the or each CDR is derived from a mammalian antibody andpreferably is derived from a murine MAb.

Suitably, the CDR-grafted antibody is produced by use of recombinant DNAtechnology.

A further method for producing a CDR-grafted antibody comprisesproviding a first DNA sequence, encoding a first antibody chain in whichthe framework regions are predominantly derived from a first antibody(acceptor) and at least one CDR is derived from a second antibody(acceptor), under the control of suitable upstream and downstreamelements; transforming a host cell with the first DNA sequence; andculturing the transformed host cell so that a CDR-grafted antibody isproduced.

Preferably, the method further comprises: providing a second DNAsequence, encoding a second antibody chain complementary to the firstchain, under the control of suitable upstream and downstream elements;and transforming the host cell with both the first and second DNAsequences.

Advantageously, the second DNA sequence encodes a second antibody chainin which the framework regions are predominantly derived from a firstantibody (acceptor) and at least one CDR is derived from the secondantibody (donor).

The first and second DNA sequences may be present on the same vector. Inthis case, the sequences may be under the control of the same ordifferent upstream and/or downstream elements.

Alternatively, the first and second DNA sequences may be present ondifferent vectors.

A nucleotide sequence may be formed which encodes an antibody chain inwhich the framework regions are predominantly derived from a firstantibody (acceptor) and at least one CDR is derived from a secondantibody (donor), the antibody chain being capable of forming aCDR-grafted antibody.

The CDR-grafted antibodies may be produced by a variety of techniques,with expression in transfected cells, such as yeast, insect, CHO ormyeloma cells, being preferred. Most preferably, the host cell is a CHOhost cell.

To design a CDR-grafted antibody, it is first necessary to ascertain thevariable domain sequence of an antibody having the desired bindingproperties. Suitable source cells for such DNA sequences include avian,mammalian or other vertebrate sources such as chickens, mice, rats andrabbits, and preferably mice. The variable domain sequences (V_(H) andV_(L)) may be determined from heavy and light chain cDNA, synthesizedfrom the respective mRNA by techniques generally known to the art. Thehypervariable regions may then be determined using the Kabat method (Wuand Kabat, J. (1970) J. Exp. Med. 132, 211). The CDRs may be determinedby structural analysis using X-ray crystallography or molecularmodelling techniques. A composite CDR may then be defined as containingall the residues in one CDR and all the residues in the correspondinghypervariable region. These composite CDRs along with certain selectresidues from the framework region are preferably transferred as the“antigen binding sites”, while the remainder of the antibody, such asthe heavy and light chain constant domains and remaining frameworkregions, may be based on human antibodies of different classes. Constantdomains may be selected to have desired effector functions appropriateto the intended use of the antibody so constructed. For example, humanIgG isotypes, IgG₁ and IgG₃ are effective for complement fixation andcell mediated lysis. For other purposes other isotypes, such as IgG₂ andIgG₄, or other classes, such as IgM and IgE, may be more suitable.

For human therapy, it is particularly desirable to use human isotypes,to minimise antiglobulin responses during therapy. Human constant domainDNA sequences, preferably in conjunction with their variable domainframework bases can be prepared in accordance with well-knownprocedures. An example of this is CAMPATH 1H available from GlaxoWellcome.

Certain CDR-grafted antibodies are provided which contain selectalterations to the human-like framework region (in other words, outsideof the CDRs of the variable domains), resulting in a CDR-graftedantibody with satisfactory binding affinity. Such binding affinity ispreferably from about 10⁵.M⁻¹ to about 10¹².M⁻¹ and is more preferablyat least about 10⁸.M⁻¹.

In constructing the CDR-grafted antibodies, the V_(H) and/or V_(L) genesegments may be altered by mutagenesis. One skilled in the art will alsounderstand that various other nucleotides coding for amino acid residuesor sequences contained in the Fc portion or other areas of the antibodymay be altered in like manner (see, for example, PCT/US89/00297).

Exemplary techniques include the addition, deletion or nonconservativesubstitution of a limited number of various nucleotides or theconservative substitution of many nucleotides, provided that the properreading frame is maintained.

Substitutions, deletions, insertions or any subcombination may be usedto arrive at a final construct. Since there are 64 possible codonsequences but only twenty known amino acids, the genetic code isdegenerate in the sense that different codons may yield the same aminoacid. Thus there is at least one codon for each amino acid, ie eachcodon yields a single amino acid and no other. It will be apparent thatduring translation, the proper reading frame must be maintained in orderto obtain the proper amino acid sequence in the polypeptide ultimatelyproduced.

Techniques for additions, deletions or substitutions at predeterminedamino acid sites having a known sequence are well known. Exemplarytechniques include oligonucleotide-mediated site-directed mutagenesisand the polymerase chain reaction.

Oligonucleotide site-directed mutagenesis in essence involveshybridizing an oligonucleotide coding for a desired mutation with asingle strand of DNA containing the region to be mutated and using thesingle strand as a template for extension of the oligonucleotide toproduce a strand containing the mutation. This technique, in variousforms, is described in Zoller and Smith (1982) Nucl. Acids Res. 10,6487.

Polymerase chain reaction (PCR) in essence involves exponentiallyamplifying DNA in vitro using sequence specific oligonucleotides. Theoligonucleotides can incorporate sequence alterations if desired. Thepolymerase chain reaction technique is described in Mullis and Fuloona(1987) Meth. Enz. 155, 335. Examples of mutagenesis using PCR aredescribed in Ho et al (1989) Gene 77, 51.

The nucleotide sequences, capable of ultimately expressing the desiredCDR-grafted antibodies, can be formed from a variety of differentpolynucleotides (genomic DNA, cDNA, RNA or synthetic oligonucleotides).At present, it is preferred that the polynucleotide sequence comprises afusion of cDNA and genomic DNA. The polynucleotide sequence may encodevarious Ig components (eg V, J, D, and C domains). They may beconstructed by a variety of different techniques. Joining appropriategenomic and cDNA sequences is presently the most common method ofproduction, but cDNA sequences may also be utilized (see EP-A-0 239400).

9. Raising an Antibody Response in a Patient

Active immunisation of the patient is preferred. In this approach, oneor more β-form PrP proteins or an aggregate thereof, especially anon-fibrillar aggregate, are prepared in an immunogenic formulationcontaining suitable adjuvants and carriers and administered to thepatient. Suitable adjuvants include Freund's complete or incompleteadjuvant, muramyl dipeptide, the “Iscoms” of EP 109 942, EP 180 564 andEP 231 039, aluminium hydroxide, saponin, DEAE-dextran, neutral oils(such as miglyol), vegetable oils (such as arachis oil), liposomes,Pluronic polyols or the Ribi adjuvant system (see, for example GB-A-2189 141). “Pluronic” is a Registered Trade Mark.

It may be advantageous to use a β-form PrP protein or an aggregatethereof from a species other than the one being treated, in order toprovide for a greater immunogenic effect, although on the other handmaturing the species may reduce the likelihood of creating anti-αPrPantibodies. Another compound can be used instead of the whole β-form PrPprotein in order to produce inhibitory antibodies in the patient. Suchother compounds may include fragments and analogues of the β-form PrPprotein.

Skilled persons will appreciate that purification of the β-form and/orβ-form binding agents, especially antibodies, can be accomplished byconventional techniques such as affinity chromatography or phagedisplay. By “β-form binding agent” we include any agent which is able tobinds preferentially the β-form rather than the α-form of a prionprotein. Purification of β-form aggregate binding agents, especiallynon-fibrillar aggregate binding agents, can also be accomplished byconventional techniques.

The binding agent is preferably an antibody or antigen binding fragmentthereof such a Fab, Fv, ScFv and Ab, but it may also be any other ligandwhich exhibits the preferential binding characteristic mentioned above.

Affinity chromatography is described in Scopes, R. K. (1993) ProteinPurification: principles and practice 3^(rd) Ed. Springer-Verlag, NewYork, ISBN 0-387-44072-3, 3-540-94072-3. (See chapters 7 and 9 inparticular).

Further information on the above affinity chromatography techniques andthe immunoassay of antigen and antibody is provided by Roitt (1991)Essential Immunology 7^(th) Ed. Blackwell Scientific Publications,London, ISBN 0-632-02877-7 (see chapter 5 in particular).

The disclosure of the above references is incorporated herein byreference. Nevertheless, an the outline of known methods is describedherein.

Purification of Antigens and Antibodies by Affinity Chromatography

Antigen or antibody is bound through its free amino groups tocyanogen-bromide-activated Sepharose particles. Insolubilized antibody,for example, can be used to pull the corresponding antigen out ofsolution in which it is present as one component of a complex mixture,by absorption to its surface. The unwanted material is washed away andthe required ligand released from the affinity absorbent by disruptionof the antigen-antibody bonds by changing the pH or adding chaotropicions such as thiocyanate. Likewise, an antigen immunosorbent can be usedto absorb out an antibody from a mixture whence it can be purified byelution. The potentially damaging effect of the eluting agent can beavoided by running the anti-serum down an affinity column so prepared asto have relatively-weak binding for the antibody being purified; underthese circumstances, the antibody is retarded in flow rate rather thanbeing firmly bound. If a protein mixture is separated by iso-electricfocusing into discrete bands, an individual band can be used to affinitypurify specific antibodies from a polyclonal antiserum.

Immunoassay of Antigen and Antibody with Labelled Reagents

Antigen and antibody can be used for the detection of each other and avariety of immunoassay techniques have been developed in which the finalread-out of the reaction involves a reagent conjugated with anappropriate label. Radiolabelling with ¹³¹I, ¹²⁵I, is an establishedtechnique.

Soluble Phase Immunoassays

Radioimmunoassay (RIA) for Antigen

The binding of radioactively labelled antigen to a limited fixed amountof antibody can be partially inhibited by addition of unlabelled antigenand the extent of this inhibition can be used as a measure of theunlabelled material added.

For Antibody

The antibody content of a serum can be assessed by the ability to bindto antigen which has been in and immobilised by physical absorption to aplastic tube or micro-agglutination tray with multiple wells; the boundimmunoglobin may then be estimated by addition of a labelled anti-Igraised for anther species. For example, a patient's serum is added to amicrowell coated with antigen, the antibodies will bind to the plasticand remaining serum proteins can be readily washed away. Bound antibodycan be estimated by addition of ¹²⁵I-labelled purified rabbit anti IgG;after rinsing out excess unbound reagent, the radioactivity of the rubewill be a measure of the antibody content of the patient's serum. Thedistribution of antibody in different classes can obviously bedetermined by using specific antisera.

Immunoradiometric Assay for Antigen

This differs from radioimmunoassay in the sense that the labelledreagent is used in excess. For the estimation of antigen, antibodies arecoated on to a solid surface such as plastic and the test antigensolution added; after washing, the amount of antigen bound to theplastic can be estimated by adding an excess of radio-labelled antibody.The specificity of the method can be improved by the sandwich assaywhich uses solid phase and labelled antibodies with specificities fordifferent parts of the antigen:

Because of health hazards and the deterioration of reagents throughradiation damage, types of label other than radiosotopes have beensought.

ELISA (Enzyme-linked Immunosorbent Assay)

Perhaps the most widespread alternative has been the use of enzymeswhich give a coloured reaction product, usually in solid phase assays.Enzymes such as horse radish peroxidase and phosphatase have been widelyemployed. A way of amplifying the phosphatase reaction is to use NADP asa substrate to generate AND which now acts as a coenzyme for a secondenzyme system. Pyrophosphatase from E.coli provides a good conjugatebecause the enzyume is not present in tissues, is stable and gives agood reaction colour. Chemi-luminescent systems based on enzymes such asluciferase can also be used.

Conjugation with the vitamin biotin is frequently used since this canreadily be detected by its reaction with enzyme-linked avidin orstreptavidin to which it binds with great specificity and affinity.

10. Identification of Ligands by Phage Display

The display of proteins and polypeptides on the surface of bacteriophage(phage), fused to one of the phage coat proteins, provides a powerfultool for the selection of specific ligands. This ‘phage display’technique was originally used by Smith in 1985 (Science 228, 1315-7) tocreate large libraries of antibodies for the purpose of selecting thosewith high affinity for a particular antigen. More recently, the methodhas been employed to present peptides, domains of proteins and intactproteins at the surface of phages in order to identify ligands havingdesired properties.

The principles behind phage display technology are as follows:

(i) Nucleic acid encoding the protein or polypeptide for display iscloned into a phage;

(ii) The cloned nucleic acid is expressed fused to the coat-anchoringpart of one of the phage coat proteins (typically the p3 or p8 coatproteins in the case of filamentous phage), such that the foreignprotein or polypeptide is displayed on the surface of the phage;

(iii) The phage displaying the protein or polypeptide with the desiredproperties is then selected (e.g. by affinity chromatography) therebyproviding a genotype (linked to a phenotype) that can be sequenced,multiplied and transferred to other expression systems.

Alternatively, the foreign protein or polypeptide may be expressed usinga phagemid vector (i.e. a vector comprising origins of replicationderived from a phage and a plasmid) that can be packaged as a singlestranded nucleic acid in a bacteriophage coat. When phagemid vectors areemployed, a “helper phage” is used to supply the functions ofreplication and packaging of the phagemid nucleic acid. The resultingphage will express both the wild type coat protein (encoded by thehelper phage) and the modified coat protein (encoded by the phagemid),whereas only the modified coat protein is expressed when a phage vectoris used.

Methods of selecting phage expressing a protein or peptide with adesired specificity are known in the art. For example, a widely usedmethod is “panning”, in which phage stocks displaying ligands areexposed to solid phase coupled target molecules, e.g. using affinitychromatography.

Alternative methods of selecting phage of interest include SAP(Selection and Amplification of Phages; as described in WO 95/16027) andSIP (Selectively-Infective Phage; EP 614989A, WO 99/07842), which employselection based on the amplification of phages in which the displayedligand specifically binds to a ligand binder. In one embodiment of theSAP method, this is achieved by using non-infectious phage andconnecting the ligand binder of interest to the N-terminal part of p3.Thus, if the ligand binder specifically binds to the displayed ligand,the otherwise non-infective ligand-expressing phage is provided with theparts of p3 needed for infection. Since this interaction is reversible,selection can then be based on kinetic parameters (see Duenas et al.,1996, Mol. Immunol. 33, 279-285).

The use of phage display to isolate ligands that bind biologicallyrelevant molecules has been reviewed in Felici et al. (1995) Biotechnol.Annual Rev. 1, 149-183, Katz (1997) Annual Rev. Biophys. Biomol. Struct.26, 27-45 and Hoogenboom et al. (1998) Immunotechnology 4(1), 1-20.Several randomised combinatorial peptide libraries have been constructedto select for polypeptides that bind different targets, e.g. cellsurface receptors or DNA (reviewed by Kay, 1995, Perspect. DrugDiscovery Des. 2, 251-268; Kay and Paul, 1996, Mol. Divers. 1, 139-140).Proteins and multimeric proteins have been successfully phage-displayedas functional molecules (see EP 0349578A, EP 0527839A, EP 0589877A;Chiswell and McCafferty, 1992, Trends Biotechnol. 10, 80-84). Inaddition, functional antibody fragments (e.g. Fab, single chain Fv[scFv]) have been expressed (McCafferty et al., 1990, Nature 348,552-554; Barbas et al., 1991, Proc. Natl. Acad. Sci. USA 88, 7978-7982;Clackson et al., 1991, Nature 352, 624-628), and some of theshortcomings of human monoclonal antibody technology have beensuperseded since human high affinity antibody fragments have beenisolated (Marks et al., 1991, J. Mol. Biol. 222, 581-597; Hoogenboom andWinter, 1992, J. Mol. Biol. 227, 381-388). Further information on theprinciples and practice of phage display is provided in Phage display ofpeptides and proteins: a laboratory manual Ed Kay, Winter and McCafferty(1996) Academic Press, Inc ISBN 0-12-402380-0, the disclosure of whichis incorporated herein by reference.

11. Immunisation—Preferred Protocols

11a. Preparation of Antigen

For the preparation of monoclonal antibodies (mAbs), β-PrP or itsderivatives may be provided in an acetate buffer as described above.Antigens may be physically (by creating recombinant β-PrP fusionproteins) or chemically coupled to suitable carrier proteins to provideadditional T cell help for immunisation in PRNP^(+/+) mice and otherrodents.

11b. Mice of various strains, rats, hamsters or rabbits can beinoculated subcutaneously with β-PrP (or an aggregate thereof,especially a non-fibrillar aggregate (50-100 μg/ animal), emulsified incomplete/incomplete Freunds adjuvant at 3 weekly intervals (Days0,20,41). At day 37 anti-peptide activity can be assayed by ELISA. Onday 48 in the case of animals used for mAb production, a finalintraperitoneal boost can be given and the animals killed for fusion 3days later (day 50). In the case of rabbits inoculated to producepolyclonal antibodies, the animals may be bled after the final boost,and at regular subsequent intervals with or without further inoculationdepending on anti-β PrP titre.

12. Monoclonal Antibody Preparation

Routine methods may be used (Galfre G., and Milstein, C. 1981 Methods inEnzymology 73, 3-46)

12a. Myeloma Cells

The following fusion partners may be used:

Mouse NSO/u Clark M. R., and Milstein, C. 1982 Somatic Cells Genetics 7,657-666 X63/Ag 8.653 Keraney et al. 1979 J. Immunol. 123, 1548-1550SP2/0 Sanchez-Madrid et al 1983 J. Immunol 130, 309-312 Bluestone 1987PNAS 84, 1374 Rat Y3 (210.RCY3.Ag 1.2.3)YO Galfre G., and Milstein, C.fusions 1981 Methods in Enzymology 73, 3-46 Hamster SP2/0 fusions

11b. Fusion Procedure

Two spleens from mice that have produced high titre antibody are fused.Myeloma cells growing in exponential phase may be mixed with splenicsingle cell suspensions in appropriate ratios, washed free of serum, andthen gently resuspended in a 50% polyethylene glycol solution at 37° C.followed after 1-2 minutes with increasing volumes of serum-free medium.After a further incubation in RPMI/10% foetal calf serum (RF₁₀) at 37°C. week and thereafter in RF10. At day 10-14 positive wells are screenedfor anti-PrP antibody by ELISA. Positive wells are then repeatedlycloned by limiting dilution until stable. Hybridomas cryopreserved inFCS 10% DMSO are stored in liquid N₂ dewars.

13. Screening for Anti-β PrP Antibodies in Serum

Recombinant PrP (0.5-10 μg/well), may be dialysed against appropriatecoating buffer (pH 4-10) and adsorbed to standard ELISA plates for 30-60minutes at 37° C. prior to washing ×4 in PBS/Tween 0.05% (PBST). Afterblocking in PBS/BSA 2% with or without additional sera, dilutions ofserum are incubated in duplicate as are relevant negative and positivecontrols. After washing, the peroxidase conjugated anti-IgG secondary isincubated, washed and then fresh ortho-phenyl diamine (OPD) substrateadded. Finally after stopping the reaction with 3M sulphuric acid theabsorbance is measured at 492 nm.

14. Screening Culture Supernatants for PrP^(Sc)-specific MonoclonalAntibodies

This may involve a staged two day procedure. On day 1, 50 μl of thegrowing cultures may be screened for anti-β PrP IgG as in the ELISAdescribed above. This β-PrP may or may not be first digested withproteinase K to remove any alpha PrP species. Positive wells in thisassay may then be screened the following day in a dot blot assaymodified from Collinge et al 1995 Lancet 346:569-570. Dot blot apparatus(ELIFA, Pierce Wariner) can be used that allows the simultaneousscreening of multiple supernatants. Supernatants can be screened forbinding to recombinant β-PrP, 1% normal human brain homogenate and to apool of 1% homogenates from CJD brains containing types 1-4, thusenabling the preferential selection of PrP^(Sc)-specific mAbs. Thus onlymAbs that bind infectious prions and not PrP^(c) from normal brain willbe expanded. Alternatively, culture supernantants can be screened forpreferential binding to either alpha or β-PrP, or to synthetic peptidesto which PrP^(Sc)-specific mAbs may bind. The 15B3 PrP^(Sc)-specific mAbcross-reacts with human, bovine and murine PrP^(Sc), and its epitope hasbeen mapped with linear synthetic peptides to three regions on thebovine PrP molecule: residues 142-148, 162-170 and 214-226 and later twoof which may not be recognised by antibodies that bind to both PrP^(c)and PrP^(Sc) (Korth C. et al. 1997 Nature 390, 74-77). These peptidesare adsorbed to ELISA plates with poly-lysine.

Monoclonal Antibodies Raised Against β-PrP

β-PrP is highly immunogenic in Prn-p^(0/0) (PrP null) mice immunisedsubcutaneously with soluble or aggregated protein emulsified in Freund'sadjuvant and splenocytes from hyperimmunised PrP null mice can bereadily fused with various fusion partners (eg NSO, NS1 murine myelomacells). One of the major advances of using β-PrP when making monoclonalantibodies is its use in screening. Previously, high throughputhybridoma screening has not been possible given the small amounts ofavailable purifiable native PrP. We have now developed a rapidPrP^(c)/PrP^(sc) discriminating ELISA screening protocol usingrecombinant PrP folded into either alpha or beta conformations. To datewe have found that some mAbs recognise only alpha PrP and othersrecognise both alpha and beta conformations. We presume thatPrP^(sc)-specific mAbs will recognise recombinant beta PrP and not alpharecombinant protein. An early rejection of alpha-only binding mAbsdramatically increases the efficiency of the screening process.Additional information regarding mAb epitopes has been obtained usingresponses to recombinant alpha and beta PrP pre-digested or not withvarying concentrations of proteinase K.

We have now produced 32 monoclonal anti-PrP antibodies using standardhybridoma technology. The majority of these mAbs recognise native alphaPrP in dot blots and on the surface of a wide variety of cells in flowcytometric analyses as well as denatured PrP derived from normal or TSEbrain homogenates.

15. Characterisation of mAbs

Immunoglobin subclass and culture supernatant Ig concentration can bemeasured by standard ELISA techniques. The fine specificity of PrP^(c)or PrP^(Sc) specific mAbs can be defined either by using a gridded arrayof overlapping human PrP peptides (synthesised commercially by JerinoBio Tools GMbH) or by using pools of PrP synthetic peptides (synthesisedindividually using standard f-moc chemistry) in the standard ELISA.Measurements of the affinity of anti-PrP mAbs for their ligands can belo made using surface plasmon resonance. Direct comparisons can be madeof mAb binding to alpha and β-PrP molecules.

16. Binding of mAbs to Surface Bound and Intracellular PrP

Flow cytometry and immunofluorescence microscopy may be used to studysurface and intracellular PrP^(c)/PrP^(Sc) expression in cell lines thatexpress surface PrP (eg EVBV lymphoblastoid, U937, K562, HEl) andperipheral blood mononuclear cells.

17. Binding to PrP in Tissue Sections

Both acetone fixed fresh frozen sections and fixed paraffin embeddedsections from normal and CJD/BSE/scrapie tissue can be used to assessthe usefulness of β-PrP binding mAbs in routine immunohistochemistry.

18. Use of Antibody in the Diagnosis of a Prion Disease

The detection of the disease-associated isoform of prion protein,PrP^(Sc), in brain or other tissues from patients is thought to bediagnostic of prion disease. To distinguish PrP^(Sc) from its cellularprecursor, PrP^(c), requires either pre-treatment with proteinase K,which will completely digest PrP^(c), but only removes aprotease-sensitive N-terminal of PrP^(Sc) or, alternatively, wouldrequire an antibody which distinguished between PrP^(c) and PrP^(Sc).Only one such selective antibody (Korth C. et al. 1997 Nature 390,74-77) has yet been reported and appears to be able to selectivelyimmunoprecipitate PrP^(Sc). It is not clear as yet, however, whetherthis antibody offers any increase in diagnostic sensitivity overexisting monoclonals. It is an IgM antibody and is likely to be of lowaffinity for PrP^(Sc). By using recombinant human PrP, and in particularthe β-form of the invention, or an aggregate thereof, especially anon-fibrillar aggregate, we should produce antibodies with highdiagnostic sensitivity as well as specificity. Anti β-PrP antibodies maybe PrP^(Sc)-specific or, alternatively, detect low levels of β-PrPmonomer in blood or other tissues or bodily fluids or materials,including faeces, urine, sputum, lymph, lymph nodes, tonsil, appendixtissue, cerebrospinal fluid, or derivatives or components thereof.

Skilled persons will appreciate that the β-form specific binding agentssuch as antibodies of the invention can be used in subtraction assayswhich involve pretreatment of a sample with a binding agent such as anantibody specific for the normal cellular α-form of a prion protein,Prp^(C), followed by treatment with a β-form specific binding agent egantibody and detection of anti β-form binding. The pre-treatment stepincreases the sensitivity of the assay for the β-form.

Similar subtraction methods are described in WO98/16834.

Many detection systems are available for using a monoclonal antibody todiagnose a disease. A number of possibilities are discussed below:

19. Detection of PrP^(Sc) in Body Fluids or Tissue Homogenates

a. Sandwich ELISA can be used to detect PrP^(Sc) in body fluids eg serumor cerebropsinal fluid (CSF). This relies on using immobilisedultrasensitive PrP^(Sc)-specific mAbs to capture PrP^(Sc) in solutionand then using biotinylated mAbs or rabbit polyclonal antiserum withspecificity for alternative PrP epitopes to detect the immobilisedcomplexes. The same techniques can be used to detect PrP^(Sc) in tissuehomogenates.

b. Dot blots may be used. Here tissue homogenates are placed directly ona suitable membrane and be treated with proteinase K to remove PrP^(c).The membrane can be incubated with anti-PrP antibodies and then suchbinding detected using an appropriate, labelled secondary antibody.Various labelling systems, involving enzymatic, fluorescent,radioisotopic or chemiluminescent methods are commonly used.

c. Standard Western blotting techniques can be used. These methods allownot only the detection of PrP, but of specific patterns of bandingfollowing proteinase K digestion. These patterns allow the recognitionof distinct strains of prions and allow, for instance, thedifferentiation of new variant CJD from classical CJD (see Collinge etal. 1996 Nature 383, 685-690 and international PCT patent applicationpublished as WO 98/16834).

d. Diagnostic methods may be developed based on the differentialaffinity of anti-PrP mAbs for PrP^(c) and PrP^(Sc). Surface plasmonresonance is ideally suited for this purpose. In such assays, purifiedanti-PrP mAbs are immobilised and binding to solubilised PrP measureddirectly from tissue fluids and homogenates. Enrichment of PrP^(Sc) bydifferential centrifugation or affinity purification may be requiredprior to the above assays.

20. Detection of Cell Associated PrP^(Sc)

It is likely that the levels of PrP^(Sc) in peripheral blood mononuclearcells (PBMC) of vCJD patients will be low and detection will depend onoptimising methods for surface and intracellular detection of PrP andthen identifying lymphocyte sub-populations with the highest prion load.Anti-β PrP mAbs can be purified and conjugated to biotin offluorochromes for this purpose. Dual and three colour flow cytometry canbe used to identify the PrP^(Sc) bearing cell types. After surfacestaining by conventional techniques, intracellular PrP can be detectedafter fixation and permeabilisation of the cell membranes. Cellularmanipulation (eg stimulation of proliferation of the pharmacologicalblockade of intracellular secretory or endocytic pathways) may be usedto enhance PrP detection.

21. Immunohistochemistry

Prion disease may be diagnosed by abnormal patterns of PrPimmunoreactivity on either formalin fixed, or frozen, tissue sectionsusing established immonohistochemical detection techniques. Frozentissue sections of whole brains (histoblots) may be treated withproteinase K and similarly exposed to antibodies to detect patterns ofPrP^(Sc) deposition which may also allow discrimination of prion straintypes.

22. Detection of Anti-PrP^(Sc) Antibodies in TSE

Although it is assumed that anti-PrP^(Sc) is not induced during thecourse of natural scrapie infection, this has not been studiedsytematicially in any form of CJD. Thus to detect anti-PrP^(Sc) we mayabsorb β-PrP to immunosorbent plates and perform standard ELISA asabove.

23. Detection of PrP Using Highly Sensitive in Vitro Lymphocyte Assays

Specific T cells are extremely sensitive to the presence of theircognate antigen. PrP-specific T cell lines/clones raised in PRNP^(0/0)mice can be used to detect PrP^(Sc) after its absorption toimmunomagnetic particles using PrP^(Sc)-specific mAbs (after Hawke et al1992 Journal of Immunological Methods 155(1):41-48). In this methodPrP^(Sc) absorbed to the particles is co-cultured with specific Tlymphocytes and antigen presenting cells and proliferation (usingstandard ³H-thymidine incorporation assays) and/or cytokine release ismeasured.

24. Toxicity of β-PrP

To examine the effect of β-PrP, in vivo, mice were inoculated withsoluble (low salt) and aggregated (200 mM NaCl) forms of the recombinantmurine protein. The recombinant, cellular PrP^(C) form was also includedin the experiment as a control.

By “low salt” we mean an ionic strength which is insufficient to causeaggregation of β-PrP, for example 0 mM to 25 mM.

The salt-treated, aggregated β-PrP material has two forms, as identifiedby electron microscopy. Addition of 200 mM NaCl causes a rapid formation(<1 hour) of spherical particles (10-20 nm diameter) and furtherincubation (>24 hours) leads to the formation of fibrillar structures.Because salt addition leads to a time-dependent change in the structureof β-PrP, three different inocula were used: low salt, short saltincubation (2-minutes) and long salt incubation (30 hours).

In order to test whether any pathological effects were dependent onexpression of PrP^(C) in the recipient, two mouse genotypes were used:TG20 (over-expressing mouse PrP) and SV129/B6 (PrP ablated).

Ablated mice are described in Beuler, H., 1992 Nature 356:577-582.

TG20 mice are described in Fischer, M., 1996 The EMBO Journal 15(6):1255-1264.

Animals were anaesthetised and inoculated intra-cranially with 30 μLaliquots of protein solution (1.6 mg/ml). After recovery from theanaesthetic some of the mice suffered immediate and severe fits and diedwithin 5 minutes. This acute toxicity was most prevalent in the TG20mice after inoculation with β-PrP which had undergone a short saltincubation. The PrP-ablated mice showed no susceptibility to β-PrP inany of its 3 forms. The results are given in the table below.

TG20 SV129/B6 (PrP^(c) over expression) (PrP ablated) PrP^(c) 0/8  N.D.β-PrP - soluble, low 4/10 0/10 salt β-PrP-200 mM NaCl 5/10 0/10 shortincubation β-PrP-200 mM 1/10 1/10 NaCl long incubation Buffer control0/10 N.D N.D. = none detected.

The toxicity of β-PrP in these circumstances is acute and therefore itcan be argued that the effect is unlike that seen in chronic T.S.E.s.However, the amount of PrP material introduced into the brain (˜50 μg)is extremely large and, more importantly, the effect is mediated byPrP^(C). Given that T.S.E.s can only infect animals which expressPrP^(C), it is likely that the effects elicited by β-PrP in thisexperiment are relevant to prion diseases. One hypothesis which isconsistent with the above observations is that the toxic agent inT.S.E.s is not the fibrillar insoluble material but a transiently formedlow molecular weight form which goes on to form these high-orderaggregates. This toxic material never reaches high steady-state levelsduring the disease and so the rate of synaptic loss and cell death isslow. When large quantities are introduced in a single dose then thereis a sudden, widespread effect on neurones which, in this initial phase,leads to sustained depolarisation and the consequent fits. The fact thatthis effect is only seen on neurones with endogenous PrP^(C) suggeststhat the effect is mediated by interactions between β-PrP and PrP^(C).In the chronic Prion diseases there is only sufficient β-PrP at any onetime to affect a small number of neurones, but long-term exposure to lowlevels of the agent leads to a slow loss of synaptic connections andeventual death of cells. We term this lethal form of the proteinβ-PrP^(L).

This represents the first occasion on which toxic Prions have been madein vitro and the results demonstrate the importance of our productionand characterisation of the soluble β-form precursor of the toxicaggregated material.

25. Identification of Compounds Capable of Inhibiting and/or ReversingConversion of a Prion Protein from its α Conformation to aβ-Conformation or from β-form to Aggregated and/or Amyloid Form,Especially a Non-fibrillar Aggregate.

Use of β-PrP in High-throughput Screening for Potential Therapeutics

The experiments thus far performed on the β-PrP structure can besummarised:

The first transition is reversible, with the β-PrP conformation beingfavoured by lowering the pH to an acidic pH, for example pH 4. Thesecond transition is effectively irreversible and results in theformation of the aggregated and/or amyloid, especially a non-fibrillaraggregate, form which scatters light owing to the large particle size.The system can be kept in the monomeric β-PrP form by maintaining a lowionic strength eg 20 mM NaCl or equivalent. When the ionic strength israised (by use of guanidinium chloride, sodium chloride, or potassiumchloride at a concentration of from 100-200 mM, especially 200 mM ormore, for instance) the system shifts towards the aggregated and/oramyloid state.

The availability and understanding of this system allows the design ofroutine and rapid assays for compounds which prevent aggregated and/oramyloid formation, especially the toxic non-fibrillar aggregatementioned in section 24.

The simplest and technically most direct method is to screen for anycompound which blocks the second transition by poising the system in theβ-PrP (reduced, monomeric) state at pH 4 and low ionic strength, forexample 200 mM NaCl. Compounds will then be added to this proteinsolution and incubated in screening wells. The next step will be toincrease the ionic strength by the addition of NaCl, KCl or similarcompound which would normally promote the formation of the aggregatedand/or amyloid form and cause an increase in light scattering in the400-500 nm range of wavelengths. Any compound, added at the first stage,which was capable of binding to and stabilising either the α-PrP(reduced, monomeric) form and/or the β-PrP (reduced, monomeric) formwill show a low scattering signal in the relevant well.

Such a system can be rapidly optimised for a high throughput screen byuse of large, multi-well microtitre plates handled by robotic systems.Screening of hundreds of thousands of different compounds is thenentirely feasible over a timescale of several months. Even larger scalescreens, of millions of compounds, is also entirely possible withallocation of sufficient technical resources. Assuming sufficientdiversity within the chemical libraries screened, it ought to bepossible to identify compounds which inhibit β-PrP or aggregated β-PrPformation at extremely low concentration, which can then be furtherevaluated.

Recombinant β-PrP: Vaccine Potential

Disruption of the transformation of normal cellular PrP is potentiallyachievable using antibodies directed at either PrP^(c) of PrP^(sc) orboth. However, it has long been recognised that anti-PrP immunity is notinduced during the course of natural TSE. This can be most readilyexplained by the widespread expression of tolerogenic levels of PrP inthe lymphoreticular system; particularly in the thymus where T cellsdevelop. Unless helper T cells are stimulated by an immunogen, B cellswill not be driven to differentiate into antibody-secreting plasmacells. It is known that physical linkage of a ‘carrier’ protein to theantibody target may overcome the need for its recognition by T cells.Despite the fact that PrP^(c) is expressed on many haemopoetic cells inthe bone marrow making tolerance of PrP-binding B cells also likely, wehave been able to conjugate carrier proteins to both recombinant alphaand beta PrP and induce anti-PrP antibodies in wild-type mice; evenusing mouse recombinant protein conjugates as immunogens. We have alsofound that T cell help can be provided by immunising mice with humanrecombinant PrP in either alpha or beta conformations. Presumably thesequence differences between mouse and human PrP are the stimulating Tcell epitopes. Both of these approaches are currently being tested fordisease modifying potential and they may form the basis oftherapeutic/preventative vaccination for CJD and other TSE.

26. Production of Compounds Comprising a Portion Capable of BindingPreferentially to the β-form of a Prion Protein and a Further EffectorPortion

In one preferred embodiment the compound comprises an effector portionwhich is directly or indirectly cytotoxic.

Methods for the preparation of compounds which possess a target-specificbinding portion and a directly, or indirectly, cytotoxic portion arewell known in the art.

For example, Bagshawe and his co-workers have disclosed (Bagshawe (1987)Br. J. Cancer 56, 531; Bagshawe et al. (1988) Br. J. Cancer 58, 700; WO88/07378) conjugated compounds comprising an antibody or part thereofand an enzyme which converts an innocuous pro-drug into a cytotoxiccompound. The cytotoxic compounds were alkylating agents, e.g. a benzoicacid mustard released from para-N-bis(2-chloroethyl)aminobenzoylglutamic acid by the action of Pseudomonas sp. CPG2 enzyme.

An alternative system using different pro-drugs has been disclosed (WO91/11201) by Epenetos and co-workers. The cytotoxic compounds werecyanogenic monosaccharides or disaccharides, such as the plant compoundamygdalin, which releases cyanide upon the action of a β-glucosidase andhydroxynitrile lyase.

In a further alternative system, the use of antibody-enzyme conjugatescontaining the enzyme alkaline phosphatase in conjunction with thepro-drug etoposide 4′-phosphate or 7-(2′aminoethyl phosphate) mitomycinor a combination thereof have been disclosed (EP 0 302 473; Senter etal., (1988) Proc. Natl. Acad. Sci. USA 85, 4842).

Another approach is the in vivo application of streptavidin conjugatedantibodies followed, after an appropriate period, by radioactive biotin(Hnatowich et al. (1988) J. Nucl. Med. 29, 1428-1434), or injection of abiotinylated mAb followed by radioactive streptavidin (Paganelli et al.(1990) Int. J. Cancer 45, 1184-1189).

Further examples of the targeting of compounds which are directly, orindirectly, cytotoxic are disclosed in PCT/GB94/00087 (EP 0 815 872 A2).

27. Exemplary Pharmaceutical Formulations of the Invention

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association the activeingredient (compound of the invention β-form of a prion protein or anaggregate thereof, or a binding agent, including antibody) with thecarrier which constitutes one or more accessory ingredients. In generalthe formulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

Whilst it is possible for an agent eg compound of the invention to beadministered alone, it is preferable to present it as a pharmaceuticalformulation, together with one or more acceptable carriers. Thecarrier(s) must be “acceptable” in the sense of being compatible withthe agent of the invention and not deleterious to the recipientsthereof. Typically, the carriers will be water or saline which will besterile and pyrogen free.

Formulations in accordance with the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant,inert diluent, preservative, disintegrant (eg sodium starch glycolate,cross-linked povidone, cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Moulded tablets may be made bymoulding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein using, for example,hydroxypropylmethylcellulose in varying proportions to provide desiredrelease profile.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouth-washes comprising the active ingredient in asuitable liquid carrier.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose or an appropriate fraction thereof, of an activeingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

The following examples illustrate pharmaceutical formulations accordingto the invention in which the active ingredient is selected from one ormore of antibodies and agents eg compounds of the invention:

EXAMPLE A Tablet

Active ingredient 100 mg Lactose 200 mg Starch  50 mgPolyvinylpyrrolidone  5 mg Magnesium stearate  4 mg 359 mg

Tablets are prepared from the foregoing ingredients by wet granulationfollowed by compression.

EXAMPLE B Ophthalmic Solution

Active ingredient 0.5 g Sodium chloride, analytical grade 0.9 gThiomersal 0.001 g Purified water to 100 ml pH adjusted to 7.5

EXAMPLE C Tablet Formulations

The following formulations A and B are prepared by wet granulation ofthe ingredients with a solution of povidone, followed by addition ofmagnesium stearate and compression.

Formulation A

mg/tablet mg/tablet (a) Active ingredient 250 250 (b) Lactose B.P. 21026 (c) Povidone B.P. 15 9 (d) Sodium Starch Glycolate 20 12 (e)Magnesium Stearate 5 3 500 300

Formulation B

mg/tablet mg/tablet (a) Active ingredient 250 250 (b) Lactose 150 — (c)Avicel PH 101 ® 60  26 (d) Povidone B.P. 15  9 (e) Sodium StarchGlycolate 20  12 (f) Magnesium Stearate 5  3 500 300

Formulation C

Active ingredient 100 Lactose 200 Starch 50 Povidone 5 Magnesiumstearate 4 359

The following formulations, D and E, are prepared by direct compressionof the admixed ingredients. The lactose used in formulation E is of thedirection compression type.

Formulation D

mg/capsule Active Ingredient 250 Pregelatinised Starch NF15 150 400

Formulation E

mg/capsule Active Ingredient 250 Lactose 150 Avicel ® 100 500

Formulation F (Controlled Release Formulation)

The formulation is prepared by wet granulation of the ingredients(below) with a solution of povidone followed by the addition ofmagnesium stearate and compression.

mg/tablet (a) Active Ingredient 500 (b) Hydroxypropylmethylcellulose 112(Methocel K4M Premium) ® (c) Lactose B.P. 53 (d) Povidone B.P.C. 28 (e)Magnesium Stearate 7 700

Drug release takes place over a period of about 6-8 hours and isgenerally complete after 12 hours.

EXAMPLE D CAPSULE FORMULATIONS Formulation A

A capsule formulation is prepared by admixing the ingredients ofFormulation D in Example C above and filling into a two-part hardgelatin capsule. Formulation B (infra) is prepared in a similar manner.

Formulation B

mg/capsule (a) Active ingredient 250 (b) Lactose B.P. 143 (c) SodiumStarch Glycolate 25 (d) Magnesium Stearate 2 420

Formulation C

mg/capsule (a) Active ingredient 250 (b) Macrogol 4000 BP 350 600

Capsules are prepared by melting the Macrogol 4000 BP, dispersing theactive ingredient in the melt and filling the melt into a two-part hardgelatin capsule.

Formulation D

mg/capsule Active ingredient 250 Lecithin 100 Arachis Oil 100 450

Capsules are prepared by dispersing the active ingredient in thelecithin and arachis oil and filling the dispersion into soft, elasticgelatin capsules.

Formulation E (Controlled Release Capsule)

The following controlled release capsule formulation is prepared byextruding ingredients a, b, and c using an extruder, followed byspheronisation of the extrudate and drying. The dried pellets are thencoated with release-controlling membrane (d) and filled into atwo-piece, hard gelatin capsule.

mg/capsule (a) Active ingredient 250 (b) Microcrystalline Cellulose 125(c) Lactose BP 125 (d) Ethyl Cellulose 13 513

EXAMPLE E Injectable Formulation

Active ingredient 0.200 g Sterile, pyrogen free phosphate buffer (pH7.0) to 10 ml

The active ingredient is dissolved in most of the phosphate buffer(35-40° C.), then made up to volume and filtered through a sterilemicropore filter into a sterile 10 ml amber glass vial (type 1) andsealed with sterile closures and overseals.

EXAMPLE F Intramuscular injection

Active ingredient 0.20 g Benzyl Alcohol 0.10 g Glucofurol 75 ® 1.45 gWater for Injection q.s. to 3.00 ml

The active ingredient is dissolved in the glycofurol. The benzyl alcoholis then added and dissolved, and water added to 3 ml. The mixture isthen filtered through a sterile micropore filter and sealed in sterile 3ml glass vials (type 1).

EXAMPLE G Syrup Suspension

Active ingredient 0.2500 g Sorbitol Solution 1.5000 g Glycerol 2.0000 gDispersible Cellulose 0.0750 g Sodium Benzoate 0.0050 g Flavour, Peach17.42.3169 0.0125 ml Purified Water q.s. to 5.0000 ml

The sodium benzoate is dissolved in a portion of the purified water andthe sorbitol solution added. The active ingredient is added anddispersed. In the glycerol is dispersed the thickener (dispersiblecellulose). The two dispersions are mixed and made up to the requiredvolume with the purified water. Further thickening is achieved asrequired by extra shearing of the suspension.

EXAMPLE H Suppository

mg/suppository Active ingredient (63 μm)*  250 Hard Fat, BP (WitepsolH15 - Dynamit Nobel) 1770 2020 *The active ingredient is used as apowder wherein at least 90% of the particles are of 63 μm diameter orless.

One fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45°C. maximum. The active ingredient is sifted through a 200 μm sieve andadded to the molten base with mixing, using a silverson fitted with acutting head, until a smooth dispersion is achieved. Maintaining themixture at 45° C., the remaining Witepsol H15 is added to the suspensionand stirred to ensure a homogenous mix. The entire suspension is passedthrough a 250 μm stainless steel screen and, with continuous stirring,is allowed to cool to 40° C. At a temperature of 38° C. to 40° C. 2.02 gof the mixture is filled into suitable plastic moulds. The suppositoriesare allowed to cool to room temperature.

EXAMPLE I Pessaries

mg/pessary Active ingredient 250 Anhydrate Dextrose 380 Potato Starch363 Magnesium Stearate 7 1000

The above ingredients are mixed directly and pessaries prepared bydirect compression of the resulting mixture.

28. Use in Medicine

The aforementioned β-form or an aggregate thereof or a binding agentincluding antibodies and other agents eg compounds of the invention or aformulation thereof may be administered in a variety of ways, fornon-limiting example, by any conventional method including oral andparenteral (eg subcutaneous or intramuscular) injection. The treatmentmay consist of a single dose or a plurality of doses over a period oftime, depending on the characteristics of the patient and/or theparticular prion disease against which the treatment is directed.

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Gly Gly Tyr 115 120 125 Met Leu Gly Ser Ala Met Ser ArgPro Ile Ile His Phe Gly Asn Asp 130 135 140 Tyr Glu Asp Arg Tyr Tyr ArgGlu Asn Met Tyr Arg Tyr Pro Asn Gln 145 150 155 160 Val Tyr Tyr Arg ProVal Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val 165 170 175 His Asp Cys ValAsn Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr 180 185 190 Thr Lys GlyGlu Asn Phe Thr Glu Thr Asp Val Lys Met Met Glu Arg 195 200 205 Val ValGlu Gln Met Cys Ile Thr Gln Tyr Glu Arg Glu Ser Gln Ala 210 215 220 TyrTyr Gln Arg Gly Ser Ser Met Val Leu Phe Ser Ser Pro Pro Val 225 230 235240 Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly 245 250 4 253PRT Gorilla gorilla 4 Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu PheVal Ala Thr Trp 1 5 10 15 Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro LysPro Gly Gly Trp Asn 20 25 30 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly SerPro Gly Gly Asn Arg 35 40 45 Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly GlnPro His Gly Gly Gly 50 55 60 Trp Gly Gln Pro His Gly Gly Gly Trp Gly GlnPro His Gly Gly Gly 65 70 75 80 Trp Gly Gln Pro His Gly Gly Gly Trp GlyGln Gly Gly Gly Thr His 85 90 95 Ser Gln Trp Asn Lys Pro Ser Lys Pro LysThr Asn Met Lys His Met 100 105 110 Ala Gly Ala Ala Ala Ala Gly Ala ValVal Gly Gly Leu Gly Gly Tyr 115 120 125 Met Leu Gly Ser Ala Met Ser ArgPro Ile Ile His Phe Gly Ser Asp 130 135 140 Tyr Glu Asp Arg Tyr Tyr ArgGlu Asn Met His Arg Tyr Pro Asn Gln 145 150 155 160 Val Tyr Tyr Arg ProMet Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val 165 170 175 His Asp Cys ValAsn Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr 180 185 190 Thr Lys GlyGlu Asn Phe Thr Glu Thr Asp Val Lys Met Met Glu Arg 195 200 205 Val ValGlu Gln Met Cys Ile Thr Gln Tyr Glu Arg Glu Ser Gln Ala 210 215 220 TyrTyr Gln Arg Gly Ser Ser Met Val Leu Phe Ser Ser Pro Pro Val 225 230 235240 Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly 245 250 5 245PRT Cercopithecus aethiops 5 Met Ala Asn Leu Gly Cys Trp Met Leu Val ValPhe Val Ala Thr Trp 1 5 10 15 Ser Asp Leu Gly Leu Cys Lys Lys Arg ProLys Pro Gly Gly Trp Asn 20 25 30 Thr Gly Gly Ser Arg Tyr Pro Gly Gln GlySer Pro Gly Gly Asn Arg 35 40 45 Tyr Pro Pro Gln Gly Gly Gly Gly Trp GlyGln Pro His Gly Gly Gly 50 55 60 Trp Gly Gln Pro His Gly Gly Gly Trp GlyGln Pro His Gly Gly Gly 65 70 75 80 Trp Gly Gln Gly Gly Gly Thr His AsnGln Trp His Lys Pro Ser Lys 85 90 95 Pro Lys Thr Ser Met Lys His Met AlaGly Ala Ala Ala Ala Gly Ala 100 105 110 Val Val Gly Gly Leu Gly Gly TyrMet Leu Gly Ser Ala Met Ser Arg 115 120 125 Pro Leu Ile His Phe Gly AsnAsp Tyr Glu Asp Arg Tyr Tyr Arg Glu 130 135 140 Asn Met Tyr Arg Tyr ProAsn Gln Val Tyr Tyr Arg Pro Val Asp Gln 145 150 155 160 Tyr Ser Asn GlnAsn Asn Phe Val His Asp Cys Val Asn Ile Thr Ile 165 170 175 Lys Gln HisThr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu 180 185 190 Thr AspVal Lys Met Met Glu Arg Val Val Glu Gln Met Cys Ile Thr 195 200 205 GlnTyr Glu Lys Glu Ser Gln Ala Tyr Tyr Gln Arg Gly Ser Ser Met 210 215 220Val Leu Phe Ser Ser Pro Pro Val Ile Leu Leu Ile Ser Phe Leu Ile 225 230235 240 Phe Leu Ile Val Gly 245 6 260 PRT Saimiri sciureus 6 Met Ala AsnLeu Gly Cys Trp Met Leu Val Leu Phe Val Ala Thr Trp 1 5 10 15 Ser AspLeu Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 Thr GlyGly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45 Tyr ProPro Gln Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp 50 55 60 Gly GlnPro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp 65 70 75 80 GlyGln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp 85 90 95 GlyGln Gly Gly Gly Thr His Asn Gln Trp Asn Lys Pro Ser Lys Pro 100 105 110Lys Thr Asn Met Lys His Met Ala Gly Ala Ala Ala Ala Gly Ala Val 115 120125 Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro 130135 140 Leu Ile His Phe Gly Asn Asp Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn145 150 155 160 Met Tyr Arg Tyr Pro Ser Gln Val Tyr Tyr Arg Pro Val AspGln Tyr 165 170 175 Ser Asn Gln Asn Asn Phe Val His Asp Cys Val Asn ValThr Ile Lys 180 185 190 Gln His Thr Val Thr Thr Thr Thr Lys Gly Glu AsnPhe Thr Glu Thr 195 200 205 Asp Val Lys Met Met Glu Arg Val Val Glu GlnMet Cys Ile Thr Gln 210 215 220 Tyr Glu Lys Glu Ser Gln Ala Tyr Tyr GlnArg Gly Ser Ser Met Val 225 230 235 240 Leu Phe Ser Ser Pro Pro Val IleLeu Leu Ile Ser Phe Leu Ile Phe 245 250 255 Leu Ile Val Gly 260 7 253PRT Macaca mulatta 7 Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu Phe ValAla Thr Trp 1 5 10 15 Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys ProGly Gly Trp Asn 20 25 30 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser ProGly Gly Asn Arg 35 40 45 Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln ProHis Gly Gly Gly 50 55 60 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln ProHis Gly Gly Gly 65 70 75 80 Trp Gly Gln Pro His Gly Gly Gly Trp Gly GlnGly Gly Gly Thr His 85 90 95 Asn Gln Trp His Lys Pro Ser Lys Pro Lys ThrSer Met Lys His Met 100 105 110 Ala Gly Ala Ala Ala Ala Gly Ala Val ValGly Gly Leu Gly Gly Tyr 115 120 125 Met Leu Gly Ser Ala Met Ser Arg ProLeu Ile His Phe Gly Asn Asp 130 135 140 Tyr Glu Asp Arg Tyr Tyr Arg GluAsn Met Tyr Arg Tyr Pro Asn Gln 145 150 155 160 Val Tyr Tyr Arg Pro ValAsp Gln Tyr Ser Asn Gln Asn Asn Phe Val 165 170 175 His Asp Cys Val AsnIle Thr Ile Lys Gln His Thr Val Thr Thr Thr 180 185 190 Thr Lys Gly GluAsn Phe Thr Glu Thr Asp Val Lys Met Met Glu Arg 195 200 205 Val Val GluGln Met Cys Ile Thr Gln Tyr Glu Lys Glu Ser Gln Ala 210 215 220 Tyr TyrGln Arg Gly Ser Ser Met Val Leu Phe Ser Ser Pro Pro Val 225 230 235 240Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly 245 250 8 253 PRTHylobates sp. 8 Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu Phe Val AlaThr Trp 1 5 10 15 Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys Pro GlyGly Trp Asn 20 25 30 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro GlyGly Asn Arg 35 40 45 Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro HisGly Gly Gly 50 55 60 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro HisGly Gly Gly 65 70 75 80 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln GlyGly Gly Thr His 85 90 95 Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr AsnMet Lys His Met 100 105 110 Ala Gly Ala Ala Ala Ala Gly Ala Val Val GlyGly Leu Gly Gly Tyr 115 120 125 Met Leu Gly Ser Ala Met Ser Arg Pro IleIle His Phe Gly Ser Asp 130 135 140 Tyr Glu Asp Arg Tyr Tyr Arg Glu AsnMet His Arg Tyr Pro Asn Gln 145 150 155 160 Val Tyr Tyr Arg Pro Met AspGln Tyr Ser Ser Gln Asn Asn Phe Val 165 170 175 His Asp Cys Val Asn IleThr Ile Lys Gln His Thr Val Thr Thr Thr 180 185 190 Thr Lys Gly Glu AsnPhe Thr Glu Thr Asp Val Lys Met Met Glu Arg 195 200 205 Val Val Glu GlnMet Cys Ile Thr Gln Tyr Glu Arg Glu Ser Gln Ala 210 215 220 Tyr Tyr GlnArg Gly Ser Ser Met Val Leu Phe Ser Ser Pro Pro Val 225 230 235 240 IleLeu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly 245 250 9 253 PRT Macacasp. 9 Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu Phe Val Ala Thr Trp 15 10 15 Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn20 25 30 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg35 40 45 Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly50 55 60 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly65 70 75 80 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly ThrHis 85 90 95 Asn Gln Trp His Lys Pro Ser Lys Pro Lys Thr Ser Met Lys HisMet 100 105 110 Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu GlyGly Tyr 115 120 125 Met Leu Gly Ser Ala Met Ser Arg Pro Leu Ile His PheGly Asn Asp 130 135 140 Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr ArgTyr Pro Asn Gln 145 150 155 160 Val Tyr Tyr Arg Pro Val Asp Gln Tyr SerAsn Gln Asn Asn Phe Val 165 170 175 His Asp Cys Val Asn Ile Thr Ile LysGln His Thr Val Thr Thr Thr 180 185 190 Thr Lys Gly Glu Asn Phe Thr GluThr Asp Val Lys Met Met Glu Arg 195 200 205 Val Val Glu Gln Met Cys IleThr Gln Tyr Glu Lys Glu Ser Gln Ala 210 215 220 Tyr Tyr Gln Arg Gly SerSer Met Val Leu Phe Ser Ser Pro Pro Val 225 230 235 240 Ile Leu Leu IleSer Phe Leu Ile Phe Leu Ile Val Gly 245 250 10 253 PRT Macaca sp. 10 MetAla Asn Leu Gly Cys Trp Met Leu Val Leu Phe Val Ala Thr Trp 1 5 10 15Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 50 55 60Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 65 70 7580 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His 85 9095 Asn Gln Trp His Lys Pro Ser Lys Pro Lys Thr Ser Met Lys His Met 100105 110 Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr115 120 125 Met Leu Gly Ser Ala Met Ser Arg Pro Leu Ile His Phe Gly AsnAsp 130 135 140 Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr ProAsn Gln 145 150 155 160 Val Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn GlnAsn Asn Phe Val 165 170 175 His Asp Cys Val Asn Ile Thr Ile Lys Gln HisThr Val Thr Thr Thr 180 185 190 Thr Lys Gly Glu Asn Phe Thr Glu Thr AspVal Lys Met Met Glu Arg 195 200 205 Val Val Glu Gln Met Cys Ile Thr GlnTyr Glu Lys Glu Ser Gln Ala 210 215 220 Tyr Tyr Gln Arg Gly Ser Ser MetVal Leu Phe Ser Ser Pro Pro Val 225 230 235 240 Ile Leu Leu Ile Ser PheLeu Ile Phe Leu Ile Val Gly 245 250 11 253 PRT Macaca sp. 11 Met Ala AsnLeu Gly Cys Trp Met Leu Val Leu Phe Val Ala Thr Trp 1 5 10 15 Ser AspLeu Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 Thr GlyGly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45 Tyr ProPro Gln Gly Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 50 55 60 Trp GlyGln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 65 70 75 80 TrpGly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His 85 90 95 AsnGln Trp His Lys Pro Ser Lys Pro Lys Thr Ser Met Lys His Met 100 105 110Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr 115 120125 Met Leu Gly Ser Ala Met Ser Arg Pro Leu Ile His Phe Gly Asn Asp 130135 140 Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln145 150 155 160 Val Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn AsnPhe Val 165 170 175 His Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr ValThr Thr Thr 180 185 190 Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Val LysMet Met Glu Arg 195 200 205 Val Val Glu Gln Met Cys Ile Thr Gln Tyr GluLys Glu Ser Gln Ala 210 215 220 Tyr Tyr Gln Arg Gly Ser Ser Met Val LeuPhe Ser Ser Pro Pro Val 225 230 235 240 Ile Leu Leu Ile Ser Phe Leu IlePhe Leu Ile Val Gly 245 250 12 253 PRT Macaca sp. 12 Met Ala Asn Leu GlyCys Trp Met Leu Val Leu Phe Val Ala Thr Trp 1 5 10 15 Ser Asp Leu GlyLeu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 Thr Gly Gly SerArg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45 Tyr Pro Pro GlnGly Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 50 55 60 Trp Gly Gln ProHis Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 65 70 75 80 Trp Gly GlnPro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His 85 90 95 Asn Gln TrpHis Lys Pro Ser Lys Pro Lys Thr Ser Met Lys His Met 100 105 110 Ala GlyAla Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr 115 120 125 MetLeu Gly Ser Ala Met Ser Arg Pro Leu Ile His Phe Gly Asn Asp 130 135 140Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln 145 150155 160 Val Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val165 170 175 His Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr ThrThr 180 185 190 Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met MetGlu Arg 195 200 205 Val Val Glu Gln Met Cys Ile Thr Gln Tyr Glu Lys GluSer Gln Ala 210 215 220 Tyr Tyr Gln Arg Gly Ser Ser Met Val Leu Phe SerSer Pro Pro Val 225 230 235 240 Ile Leu Leu Ile Ser Phe Leu Ile Phe LeuIle Val Gly 245 250 13 252 PRT Callithrix sp. 13 Met Ala Asn Leu Gly CysTrp Met Leu Phe Leu Phe Val Ala Thr Trp 1 5 10 15 Ser Asp Leu Gly LeuCys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 Thr Gly Gly Ser ArgTyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45 Tyr Pro Pro Gln GlyGly Gly Trp Gly Gln Pro His Gly Gly Gly Trp 50 55 60 Gly Gln Pro His GlyGly Gly Trp Gly Gln Pro His Gly Gly Gly Trp 65 70 75 80 Gly Gln Pro HisGly Gly Gly Trp Gly Gln Gly Gly Gly Thr His Ser 85 90 95 Gln Trp Asn LysPro Ser Lys Pro Lys Thr Asn Met Lys His Val Ala 100 105 110 Gly Ala AlaAla Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met 115 120 125 Leu GlySer Ala Met Ser Arg Pro Leu Ile His Phe Gly Asn Asp Tyr 130 135 140 GluAsp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln Val 145 150 155160 Tyr Tyr Arg Pro Val Asp Gln Tyr Asn Asn Gln Asn Asn Phe Val His 165170 175 Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr Thr180 185 190 Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met Glu ArgVal 195 200 205 Val Glu Gln Met Cys Ile Thr Gln Tyr Glu Lys Glu Ser GlnAla Tyr 210 215 220 Tyr Gln Arg Gly Ser Ser Met Val Leu Phe Ser Ser ProPro Val Ile 225 230 235 240 Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile ValGly 245 250 14 253 PRT Papio hamadryas 14 Met Ala Asn Leu Gly Cys TrpMet Leu Val Leu Phe Val Ala Thr Trp 1 5 10 15 Ser Asp Leu Gly Leu CysLys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 Thr Gly Gly Ser Arg TyrPro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45 Tyr Pro Pro Gln Gly GlyGly Gly Trp Gly Gln Pro His Gly Gly Gly 50 55 60 Trp Gly Gln Pro His GlyGly Gly Trp Gly Gln Pro His Gly Gly Gly 65 70 75 80 Trp Gly Gln Pro HisGly Gly Gly Trp Gly Gln Gly Gly Gly Thr His 85 90 95 Asn Gln Trp His LysPro Ser Lys Pro Lys Thr Ser Met Lys His Met 100 105 110 Ala Gly Ala AlaAla Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr 115 120 125 Met Leu GlySer Ala Met Ser Arg Pro Leu Ile His Phe Gly Asn Asp 130 135 140 Tyr GluAsp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln 145 150 155 160Val Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val 165 170175 His Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr 180185 190 Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met Glu Arg195 200 205 Val Val Glu Gln Met Cys Ile Thr Gln Tyr Glu Lys Glu Ser GlnAla 210 215 220 Tyr Tyr Gln Arg Gly Ser Ser Met Val Leu Phe Ser Ser ProPro Val 225 230 235 240 Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile ValGly 245 250 15 245 PRT Cercopithecus diana 15 Met Ala Asn Leu Gly CysTrp Met Leu Val Val Phe Val Ala Thr Trp 1 5 10 15 Ser Asp Leu Gly LeuCys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 Thr Gly Gly Ser ArgTyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45 Tyr Pro Pro Gln GlyGly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 50 55 60 Trp Gly Gln Pro HisGly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 65 70 75 80 Trp Gly Gln GlyGly Gly Thr His Asn Gln Trp His Lys Pro Ser Lys 85 90 95 Pro Lys Thr SerMet Lys His Met Ala Gly Ala Ala Ala Ala Gly Ala 100 105 110 Val Val GlyGly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg 115 120 125 Pro LeuIle His Phe Gly Asn Asp Tyr Glu Asp Arg Tyr Tyr Arg Glu 130 135 140 AsnMet Tyr Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp Gln 145 150 155160 Tyr Ser Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Ile Thr Ile 165170 175 Lys Gln His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu180 185 190 Thr Asp Val Lys Met Met Glu Arg Val Val Glu Gln Met Cys IleThr 195 200 205 Gln Tyr Glu Lys Glu Ser Gln Ala Tyr Tyr Gln Arg Gly SerSer Met 210 215 220 Val Leu Phe Ser Ser Pro Pro Val Ile Leu Leu Ile SerPhe Leu Ile 225 230 235 240 Phe Leu Ile Val Gly 245 16 253 PRT Colobusguereza 16 Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu Phe Val Ala ThrTrp 1 5 10 15 Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly GlyTrp Asn 20 25 30 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly GlyAsn Arg 35 40 45 Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His GlyGly Gly 50 55 60 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His GlyGly Gly 65 70 75 80 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly GlyGly Thr His 85 90 95 Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Ser MetLys His Met 100 105 110 Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly GlyLeu Gly Gly Tyr 115 120 125 Met Leu Gly Ser Ala Met Ser Arg Pro Leu IleHis Phe Gly Asn Asp 130 135 140 Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn MetTyr Arg Tyr Pro Asn Gln 145 150 155 160 Val Tyr Tyr Arg Pro Val Asp GlnTyr Ser Asn Gln Asn Asn Phe Val 165 170 175 His Asp Cys Val Asn Ile ThrIle Lys Gln His Thr Val Thr Thr Thr 180 185 190 Thr Lys Gly Glu Asn PheThr Glu Thr Asp Val Lys Met Met Glu Arg 195 200 205 Val Val Glu Gln MetCys Ile Thr Gln Tyr Glu Lys Glu Ser Gln Ala 210 215 220 Tyr Tyr Gln ArgGly Ser Ser Met Val Leu Phe Ser Ser Pro Pro Val 225 230 235 240 Ile LeuLeu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly 245 250 17 252 PRT Cebus sp.17 Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu Phe Val Ala Thr Trp 1 510 15 Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 2025 30 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Leu 3540 45 Tyr Pro Pro Gln Gly Gly Gly Trp Gly Gln Pro His Gly Gly Ser Trp 5055 60 Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp 6570 75 80 Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His Asn85 90 95 Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Ser Met Lys His Val Ala100 105 110 Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly TyrMet 115 120 125 Leu Gly Ser Ala Met Ser Arg Pro Leu Ile His Phe Gly AsnAsp Tyr 130 135 140 Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr ProAsn Gln Val 145 150 155 160 Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn GlnAsn Asn Phe Val His 165 170 175 Asp Cys Val Asn Ile Thr Ile Lys Gln HisThr Val Thr Thr Thr Thr 180 185 190 Lys Gly Glu Asn Phe Thr Glu Thr AspVal Lys Met Met Glu Arg Val 195 200 205 Val Glu Gln Met Cys Ile Thr GlnTyr Glu Arg Glu Ser Gln Ala Tyr 210 215 220 Tyr Gln Arg Gly Ser Ser MetVal Leu Phe Ser Ser Pro Pro Val Ile 225 230 235 240 Leu Leu Ile Ser PheLeu Ile Phe Leu Ile Val Gly 245 250 18 253 PRT Presbytis francoisi 18Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu Phe Val Ala Thr Trp 1 5 1015 Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 2530 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 4045 Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 50 5560 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 65 7075 80 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His 8590 95 Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Ser Met Lys His Met100 105 110 Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly GlyTyr 115 120 125 Met Leu Gly Ser Ala Met Ser Arg Pro Leu Ile His Phe GlyAsn Asp 130 135 140 Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg TyrPro Asn Gln 145 150 155 160 Val Tyr Tyr Arg Pro Val Asp Gln Tyr Ser AsnGln Asn Asn Phe Val 165 170 175 His Asp Cys Val Asn Ile Thr Ile Lys GlnHis Thr Val Thr Thr Thr 180 185 190 Thr Lys Gly Glu Asn Phe Thr Glu ThrAsp Val Lys Met Met Glu Arg 195 200 205 Val Val Glu Gln Met Cys Ile ThrGln Tyr Glu Lys Glu Ser Gln Ala 210 215 220 Tyr Tyr Gln Arg Gly Ser SerMet Val Phe Phe Ser Ser Pro Pro Val 225 230 235 240 Ile Leu Leu Ile SerPhe Leu Ile Phe Leu Ile Val Gly 245 250 19 253 PRT Hylobates syndactylus19 Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu Phe Val Ala Thr Trp 1 510 15 Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 2025 30 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 3540 45 Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 5055 60 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 6570 75 80 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His85 90 95 Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Met Lys His Met100 105 110 Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly GlyTyr 115 120 125 Met Leu Gly Ser Ala Met Ser Arg Pro Ile Ile His Phe GlySer Asp 130 135 140 Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met His Arg TyrPro Asn Gln 145 150 155 160 Val Tyr Tyr Arg Pro Met Asp Gln Tyr Ser SerGln Asn Asn Phe Val 165 170 175 His Asp Cys Val Asn Ile Thr Ile Lys GlnHis Thr Val Thr Thr Thr 180 185 190 Thr Lys Gly Glu Asn Phe Thr Glu ThrAsp Val Lys Met Met Glu Arg 195 200 205 Val Val Glu Gln Met Cys Ile ThrGln Tyr Glu Arg Glu Ser Gln Ala 210 215 220 Tyr Tyr Gln Arg Gly Ser SerMet Val Leu Phe Ser Ser Pro Pro Val 225 230 235 240 Ile Leu Leu Ile SerPhe Leu Ile Phe Leu Ile Val Gly 245 250 20 254 PRT Mus sp. 20 Met AlaAsn Leu Gly Tyr Trp Leu Leu Ala Leu Phe Val Thr Met Trp 1 5 10 15 ThrAsp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 ThrGly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45 TyrPro Pro Gln Gly Thr Gly Trp Gly Gln Pro His Gly Gly Ser Trp 50 55 60 GlyGln Pro His Gly Gly Ser Trp Gly Gln Pro His Gly Gly Gly Trp 65 70 75 80Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His Asn 85 90 95Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Leu Lys His Val Ala 100 105110 Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met 115120 125 Leu Gly Ser Ala Val Ser Arg Pro Met Ile His Phe Gly Asn Asp Trp130 135 140 Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn GlnVal 145 150 155 160 Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn AsnPhe Val His 165 170 175 Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr ValThr Thr Thr Thr 180 185 190 Lys Gly Glu Asn Phe Thr Glu Thr Asp Val LysMet Met Glu Arg Val 195 200 205 Val Glu Gln Met Cys Val Thr Gln Tyr GlnLys Glu Ser Gln Ala Tyr 210 215 220 Tyr Asp Gly Arg Arg Ser Ser Ser ThrVal Leu Phe Ser Ser Pro Pro 225 230 235 240 Val Ile Leu Leu Ile Ser PheLeu Ile Phe Leu Ile Val Gly 245 250 21 254 PRT Mus sp. 21 Met Ala AsnLeu Gly Tyr Trp Leu Leu Ala Leu Phe Val Thr Met Trp 1 5 10 15 Thr AspVal Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 Thr GlyGly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45 Tyr ProPro Gln Gly Thr Gly Trp Gly Gln Pro His Gly Gly Ser Trp 50 55 60 Gly GlnPro His Gly Gly Ser Trp Gly Gln Pro His Gly Gly Gly Trp 65 70 75 80 GlyGln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His Asn 85 90 95 GlnTrp Asn Lys Pro Ser Lys Pro Lys Thr Asn Leu Lys His Val Ala 100 105 110Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met 115 120125 Leu Gly Ser Ala Met Ser Arg Pro Met Ile His Phe Gly Asn Asp Trp 130135 140 Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln Val145 150 155 160 Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn Asn PheVal His 165 170 175 Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val ThrThr Thr Thr 180 185 190 Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys MetMet Glu Arg Val 195 200 205 Val Glu Gln Met Cys Val Thr Gln Tyr Gln LysGlu Ser Gln Ala Tyr 210 215 220 Tyr Asp Gly Arg Arg Ser Ser Ser Thr ValLeu Phe Ser Ser Pro Pro 225 230 235 240 Val Ile Leu Leu Ile Ser Phe LeuIle Phe Leu Ile Val Gly 245 250 22 254 PRT Mus sp. 22 Met Ala Asn LeuGly Tyr Trp Leu Leu Ala Leu Phe Val Thr Met Trp 1 5 10 15 Thr Asp ValGly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 Thr Gly GlySer Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45 Tyr Pro ProGln Gly Thr Gly Trp Gly Gln Pro His Gly Gly Ser Trp 50 55 60 Gly Gln ProHis Gly Gly Ser Trp Gly Gln Pro His Gly Gly Gly Trp 65 70 75 80 Gly GlnPro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His Asn 85 90 95 Gln TrpAsn Lys Pro Ser Lys Pro Lys Thr Asn Phe Lys His Val Ala 100 105 110 GlyAla Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met 115 120 125Leu Gly Ser Ala Met Ser Arg Pro Met Ile His Phe Gly Asn Asp Trp 130 135140 Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln Val 145150 155 160 Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn Asn Phe ValHis 165 170 175 Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Val ThrThr Thr 180 185 190 Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met MetGlu Arg Val 195 200 205 Val Glu Gln Met Cys Val Thr Gln Tyr Gln Lys GluSer Gln Ala Tyr 210 215 220 Tyr Asp Gly Arg Arg Ser Ser Ser Thr Val LeuPhe Ser Ser Pro Pro 225 230 235 240 Val Ile Leu Leu Ile Ser Phe Leu IlePhe Leu Ile Val Gly 245 250 23 254 PRT Mesocricetus auratus 23 Met AlaAsn Leu Ser Tyr Trp Leu Leu Ala Leu Phe Val Ala Thr Trp 1 5 10 15 ThrAsp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30 ThrGly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45 TyrPro Pro Gln Gly Gly Thr Gly Trp Gly Gln Pro His Gly Gly Gly 50 55 60 TrpGly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 65 70 75 80Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His 85 90 95Asn Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Met Lys His Val 100 105110 Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr 115120 125 Met Leu Gly Ser Ala Met Ser Arg Pro Met Leu His Phe Gly Asn Asp130 135 140 Trp Glu Asp Arg Tyr Tyr Arg Glu Asn Met Asn Arg Tyr Pro AsnGln 145 150 155 160 Val Tyr Tyr Arg Pro Val Asp Gln Tyr Asn Asn Gln AsnAsn Phe Val 165 170 175 His Asp Cys Val Asn Ile Thr Ile Lys Gln His ThrVal Thr Thr Thr 180 185 190 Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp ValLys Met Met Glu Arg 195 200 205 Val Val Glu Gln Met Cys Val Thr Gln TyrGln Lys Glu Ser Gln Ala 210 215 220 Tyr Tyr Asp Gly Arg Arg Ser Ser AlaVal Leu Phe Ser Ser Pro Pro 225 230 235 240 Val Ile Leu Leu Ile Ser PheLeu Ile Phe Leu Ile Val Gly 245 250 24 264 PRT Bos taurus 24 Met Val LysSer His Ile Gly Ser Trp Met Leu Val Leu Phe Val Ala 1 5 10 15 Met TrpSer Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly 20 25 30 Gly TrpAsn Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly 35 40 45 Gly AsnArg Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His 50 55 60 Gly GlyGly Trp Gly Gln Pro His Gly Gly Gly Gly Trp Gly Gln Pro 65 70 75 80 HisGly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro 85 90 95 HisGly Gly Gly Trp Gly Gln Gly Gly Thr Gly Asn Gln Trp Asn Lys 100 105 110Pro Ser Lys Pro Lys Thr Asn Met Lys His Val Ala Gly Ala Ala Ala 115 120125 Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala 130135 140 Met Ser Arg Pro Leu Ile His Phe Gly Ser Asp Tyr Glu Asp Arg Tyr145 150 155 160 Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln Val Tyr TyrArg Pro 165 170 175 Val Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val His AspCys Val Asn 180 185 190 Ile Thr Val Lys Glu His Thr Val Thr Thr Thr ThrLys Gly Glu Asn 195 200 205 Phe Thr Glu Thr Asp Ile Lys Met Met Glu ArgVal Val Glu Gln Met 210 215 220 Cys Ile Thr Gln Tyr Gln Arg Glu Ser GlnAla Tyr Tyr Gln Arg Gly 225 230 235 240 Ala Ser Val Ile Leu Phe Ser SerPro Pro Val Ile Leu Leu Ile Ser 245 250 255 Phe Leu Ile Phe Leu Ile ValGly 260 25 256 PRT Ovis sp. 25 Met Val Lys Ser His Ile Gly Ser Trp MetLeu Val Leu Phe Val Ala 1 5 10 15 Met Trp Ser Asp Val Gly Leu Cys LysLys Arg Pro Lys Pro Gly Gly 20 25 30 Gly Trp Asn Thr Gly Gly Ser Arg TyrPro Gly Gln Gly Ser Pro Gly 35 40 45 Gly Asn Arg Tyr Pro Pro Gln Gly GlyGly Gly Trp Gly Gln Pro His 50 55 60 Gly Gly Gly Gly Trp Gly Gln Pro HisGly Gly Gly Trp Gly Gln Pro 65 70 75 80 His Gly Gly Gly Trp Gly Gln ProHis Gly Gly Gly Trp Gly Gln Gly 85 90 95 Gly Ser His Ser Gln Trp Asn LysPro Ser Lys Pro Lys Thr Asn Met 100 105 110 Lys His Val Ala Gly Ala AlaAla Ala Gly Ala Val Val Gly Gly Leu 115 120 125 Gly Gly Tyr Met Leu GlySer Ala Met Ser Arg Pro Leu Ile His Phe 130 135 140 Gly Asn Asp Tyr GluAsp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr 145 150 155 160 Pro Asn GlnVal Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn 165 170 175 Asn PheVal His Asp Cys Val Asn Ile Thr Val Lys Gln His Thr Val 180 185 190 ThrThr Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Ile Lys Ile 195 200 205Met Glu Arg Val Val Glu Gln Met Cys Ile Thr Gln Tyr Gln Arg Glu 210 215220 Ser Gln Ala Tyr Tyr Gln Arg Gly Ala Ser Val Ile Leu Phe Ser Ser 225230 235 240 Pro Pro Val Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile ValGly 245 250 255 26 256 PRT Antilocapra americana 26 Met Val Lys Ser HisIle Gly Ser Trp Met Leu Val Leu Phe Val Ala 1 5 10 15 Met Trp Ser AspVal Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly 20 25 30 Gly Trp Asn ThrGly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly 35 40 45 Gly Asn Arg TyrPro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His 50 55 60 Gly Gly Gly GlyTrp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro 65 70 75 80 His Gly GlyGly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly 85 90 95 Gly Thr HisSer Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Met 100 105 110 Lys HisVal Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu 115 120 125 GlyGly Tyr Met Leu Gly Ser Ala Met Asn Arg Pro Leu Ile His Phe 130 135 140Gly Asn Asp Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr 145 150155 160 Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp Gln Tyr Asn Asn Gln Asn165 170 175 Thr Phe Val His Asp Cys Val Asn Ile Thr Val Lys Gln His ThrVal 180 185 190 Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp IleLys Met 195 200 205 Met Glu Arg Val Val Glu Gln Met Cys Ile Thr Gln TyrGln Arg Glu 210 215 220 Ser Gln Ala Tyr Tyr Gln Arg Gly Ala Ser Val IleLeu Phe Ser Ser 225 230 235 240 Pro Pro Val Ile Leu Leu Ile Ser Phe LeuIle Phe Leu Ile Val Gly 245 250 255 27 264 PRT Tragelaphus strepsiceros27 Met Val Lys Ser His Ile Gly Ser Trp Met Leu Val Leu Phe Val Ala 1 510 15 Met Trp Ser Asp Val Ala Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly 2025 30 Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly 3540 45 Gly Asn Arg Tyr Pro Ser Gln Gly Gly Gly Gly Trp Gly Gln Pro His 5055 60 Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Gly Trp Gly Gln Pro 6570 75 80 His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro85 90 95 His Gly Gly Gly Trp Gly Gln Gly Gly Thr His Gly Gln Trp Asn Lys100 105 110 Pro Ser Lys Pro Lys Thr Asn Met Lys His Val Ala Gly Ala AlaAla 115 120 125 Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu GlySer Ala 130 135 140 Met Ser Arg Pro Leu Ile His Phe Gly Ser Asp Tyr GluAsp Arg Tyr 145 150 155 160 Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn GlnVal Tyr Tyr Arg Pro 165 170 175 Val Asp Gln Tyr Ser Asn Gln Asn Asn PheVal His Asp Cys Val Asn 180 185 190 Ile Thr Val Lys Gln His Thr Val ThrThr Thr Thr Lys Gly Glu Asn 195 200 205 Phe Thr Glu Thr Asp Ile Lys MetMet Glu Arg Val Val Glu Gln Met 210 215 220 Cys Ile Thr Gln Tyr Gln ArgGlu Ser Glu Ala Tyr Tyr Gln Arg Gly 225 230 235 240 Ala Ser Val Ile LeuPhe Ser Ser Pro Pro Val Ile Leu Leu Ile Ser 245 250 255 Phe Leu Ile PheLeu Ile Val Gly 260 28 256 PRT Capra hircus 28 Met Val Lys Ser His IleGly Ser Trp Met Leu Val Leu Phe Val Ala 1 5 10 15 Met Trp Ser Asp ValGly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly 20 25 30 Gly Trp Asn Thr GlyGly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly 35 40 45 Gly Asn Arg Tyr ProPro Gln Gly Gly Gly Gly Trp Gly Gln Pro His 50 55 60 Gly Gly Gly Gly TrpGly Gln Pro His Gly Gly Gly Trp Gly Gln Pro 65 70 75 80 His Gly Gly GlyTrp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly 85 90 95 Gly Ser His SerGln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Met 100 105 110 Lys His ValAla Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu 115 120 125 Gly GlyTyr Met Leu Gly Ser Ala Met Ser Arg Pro Leu Ile His Phe 130 135 140 GlyAsn Asp Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr 145 150 155160 Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn 165170 175 Asn Phe Val His Asp Cys Val Asn Ile Thr Val Lys Gln His Thr Val180 185 190 Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Ile LysIle 195 200 205 Met Glu Arg Val Val Glu Gln Met Cys Ile Thr Gln Tyr GlnArg Glu 210 215 220 Ser Gln Ala Tyr Tyr Gln Arg Gly Ala Ser Val Ile LeuPhe Ser Pro 225 230 235 240 Pro Pro Val Ile Leu Leu Ile Ser Phe Leu IlePhe Leu Ile Val Gly 245 250 255 29 257 PRT Sus sp. 29 Met Val Lys SerHis Ile Gly Gly Trp Met Leu Val Leu Phe Val Ala 1 5 10 15 Ala Trp SerAsp Ile Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly 20 25 30 Gly Trp AsnThr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly 35 40 45 Gly Asn ArgTyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His 50 55 60 Gly Gly GlyGly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro 65 70 75 80 His GlyGly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly 85 90 95 Gly GlySer His Gly Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn 100 105 110 MetLys His Val Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly 115 120 125Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro Leu Ile His 130 135140 Phe Gly Ser Asp Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg 145150 155 160 Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp Gln Tyr Ser AsnGln 165 170 175 Asn Ser Phe Val His Asp Cys Val Asn Ile Thr Val Lys GlnHis Thr 180 185 190 Val Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu ThrAsp Val Lys 195 200 205 Met Ile Glu Arg Val Val Glu Gln Met Cys Ile ThrGln Tyr Gln Lys 210 215 220 Glu Tyr Glu Ala Tyr Ala Gln Arg Gly Ala SerVal Ile Leu Phe Ser 225 230 235 240 Ser Pro Pro Val Ile Leu Leu Ile SerPhe Leu Ile Phe Leu Ile Val 245 250 255 Gly 30 257 PRT Mustela sp. 30Met Val Lys Ser His Ile Gly Ser Trp Leu Leu Val Leu Phe Val Ala 1 5 1015 Thr Trp Ser Asp Ile Gly Phe Cys Lys Lys Arg Pro Lys Pro Gly Gly 20 2530 Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly 35 4045 Gly Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His 50 5560 Gly Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro 65 7075 80 His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly 8590 95 Gly Gly Ser His Gly Gln Trp Gly Lys Pro Ser Lys Pro Lys Thr Asn100 105 110 Met Lys His Val Ala Gly Ala Ala Ala Ala Gly Ala Val Val GlyGly 115 120 125 Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro LeuIle His 130 135 140 Phe Gly Asn Asp Tyr Glu Asp Arg Tyr Tyr Arg Glu AsnMet Tyr Arg 145 150 155 160 Tyr Pro Asn Gln Val Tyr Tyr Lys Pro Val AspGln Tyr Ser Asn Gln 165 170 175 Asn Asn Leu Val His Asp Cys Val Asn IleThr Val Lys Gln His Thr 180 185 190 Val Thr Thr Thr Thr Lys Gly Glu AsnPhe Thr Glu Thr Asp Met Lys 195 200 205 Ile Met Glu Arg Val Val Glu GlnMet Cys Val Thr Gln Tyr Gln Gln 210 215 220 Glu Ser Glu Ala Tyr Tyr GlnArg Gly Ala Ser Ala Ile Leu Phe Ser 225 230 235 240 Pro Pro Pro Val IleLeu Leu Ile Ser Leu Leu Ile Leu Leu Ile Val 245 250 255 Gly 31 256 PRTCanis familiaris 31 Met Val Lys Ser His Ile Gly Gly Trp Ile Leu Leu PheVal Ala Thr 1 5 10 15 Trp Ser Asp Val Gly Leu Cys Lys Lys Arg Pro LysPro Gly Gly Trp 20 25 30 Asn Thr Gly Gly Gly Ser Arg Tyr Pro Gly Gln GlySer Pro Gly Gly 35 40 45 Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp GlyGln Pro His Gly 50 55 60 Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly TrpGly Gln Pro His 65 70 75 80 Gly Gly Gly Trp Gly Gln Pro His Gly Gly GlyTrp Gly Gln Gly Gly 85 90 95 Gly Ser His Ser Gln Trp Gly Lys Pro Asn LysPro Lys Thr Asn Met 100 105 110 Lys His Val Ala Gly Ala Ala Ala Ala GlyAla Val Val Gly Gly Leu 115 120 125 Gly Gly Tyr Met Leu Gly Ser Ala MetSer Arg Pro Leu Ile His Phe 130 135 140 Gly Asn Asp Tyr Glu Asp Arg TyrTyr Arg Glu Asn Met Tyr Arg Tyr 145 150 155 160 Pro Glu Gln Val Tyr TyrPro Pro Val Asp Gln Tyr Ser Asn Gln Asn 165 170 175 Asn Phe Val Arg AspCys Val Asn Ile Thr Val Lys Gln His Thr Val 180 185 190 Thr Thr Thr ThrLys Gly Glu Asn Phe Thr Glu Thr Asp Met Lys Ile 195 200 205 Met Glu ArgVal Val Glu Gln Met Cys Val Thr Gln Tyr Gln Lys Glu 210 215 220 Ser GluAla Tyr Tyr Gln Arg Gly Ala Ser Ala Ile Leu Phe Ser Pro 225 230 235 240Pro Pro Val Ile Leu Leu Ile Ser Leu Leu Ile Leu Leu Ile Val Gly 245 250255 32 252 PRT Oryctolagus cuniculus 32 Met Ala His Leu Gly Tyr Trp MetLeu Leu Leu Phe Val Ala Thr Trp 1 5 10 15 Ser Asp Val Gly Leu Cys LysLys Arg Pro Lys Pro Gly Gly Gly Trp 20 25 30 Asn Thr Gly Gly Ser Arg TyrPro Gly Gln Ser Ser Pro Gly Gly Asn 35 40 45 Arg Tyr Pro Pro Gln Gly GlyGly Trp Gly Gln Pro His Gly Gly Gly 50 55 60 Trp Gly Gln Pro His Gly GlyGly Trp Gly Gln Pro His Gly Gly Gly 65 70 75 80 Trp Gly Gln Pro His GlyGly Gly Trp Gly Gln Gly Gly Thr His Asn 85 90 95 Gln Trp Gly Lys Pro SerLys Pro Lys Thr Ser Met Lys His Val Ala 100 105 110 Gly Ala Ala Ala AlaGly Ala Val Val Gly Gly Leu Gly Gly Tyr Met 115 120 125 Leu Gly Ser AlaMet Ser Arg Pro Leu Ile His Phe Gly Asn Asp Tyr 130 135 140 Glu Asp ArgTyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln Val 145 150 155 160 TyrTyr Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn Ser Phe Val His 165 170 175Asp Cys Val Asn Ile Thr Val Lys Gln His Thr Val Thr Thr Thr Thr 180 185190 Lys Gly Glu Asn Phe Thr Glu Thr Asp Ile Lys Ile Met Glu Arg Val 195200 205 Val Glu Gln Met Cys Ile Thr Gln Tyr Gln Gln Glu Ser Gln Ala Ala210 215 220 Tyr Gln Arg Ala Ala Gly Val Val Leu Phe Ser Ser Pro Pro ValIle 225 230 235 240 Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly 245250 33 259 PRT Trichosurus vulpecula 33 Met Gly Lys Ile Gln Leu Gly TyrTrp Ile Leu Val Leu Phe Ile Val 1 5 10 15 Thr Trp Ser Asp Leu Gly LeuCys Lys Lys Pro Lys Pro Arg Pro Gly 20 25 30 Gly Gly Trp Asn Ser Gly GlySer Asn Arg Tyr Pro Gly Gln Pro Gly 35 40 45 Ser Pro Gly Gly Asn Arg TyrPro Gly Trp Gly His Pro Gln Gly Gly 50 55 60 Gly Thr Asn Trp Gly Gln ProHis Pro Gly Gly Ser Asn Trp Gly Gln 65 70 75 80 Pro His Pro Gly Gly SerSer Trp Gly Gln Pro His Gly Gly Ser Asn 85 90 95 Trp Gly Gln Gly Gly TyrAsn Lys Trp Lys Pro Asp Lys Pro Lys Thr 100 105 110 Asn Leu Lys His ValAla Gly Ala Ala Ala Ala Gly Ala Val Val Gly 115 120 125 Gly Leu Gly GlyTyr Met Leu Gly Ser Ala Met Ser Arg Pro Val Ile 130 135 140 His Phe GlyAsn Glu Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Gln Tyr 145 150 155 160 ArgTyr Pro Asn Gln Val Met Tyr Arg Pro Ile Asp Gln Tyr Ser Ser 165 170 175Gln Asn Asn Phe Val His Asp Cys Val Asn Ile Thr Val Lys Gln His 180 185190 Thr Thr Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Ile 195200 205 Lys Ile Met Glu Arg Val Val Glu Gln Met Cys Ile Thr Gln Tyr Gln210 215 220 Ala Glu Tyr Glu Ala Ala Ala Gln Arg Ala Tyr Asn Met Ala PhePhe 225 230 235 240 Ser Ala Pro Pro Val Thr Leu Leu Phe Leu Ser Phe LeuIle Phe Leu 245 250 255 Ile Val Ser 34 277 PRT Gallus gallus 34 Met ProAla Ala Met Ala Arg Leu Leu Thr Thr Cys Cys Leu Leu Ala 1 5 10 15 LeuLeu Leu Ala Ala Cys Thr Asp Val Ala Leu Ser Lys Lys Gly Lys 20 25 30 GlyLys Pro Ser Gly Gly Gly Trp Gly Ala Gly Ser His Arg Gln Pro 35 40 45 SerTyr Pro Arg Gln Pro Gly Tyr Pro His Asn Pro Gly Tyr Pro His 50 55 60 AsnPro Gly Tyr Pro His Asn Pro Gly Tyr Pro His Asn Pro Gly Tyr 65 70 75 80Pro His Asn Pro Gly Tyr Pro Gln Asn Pro Gly Tyr Pro His Asn Pro 85 90 95Gly Tyr Pro Gly Trp Gly Gln Gly Tyr Asn Pro Ser Ser Gly Gly Ser 100 105110 Tyr His Asn Gln Lys Pro Trp Lys Pro Pro Lys Thr Asn Phe Lys His 115120 125 Val Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly130 135 140 Tyr Ala Met Gly Arg Val Met Ser Gly Met Asn Tyr His Phe AspSer 145 150 155 160 Pro Asp Glu Tyr Arg Trp Trp Ser Glu Asn Ser Ala ArgTyr Pro Asn 165 170 175 Arg Val Tyr Tyr Arg Asp Tyr Ser Ser Pro Val ProGln Asp Val Phe 180 185 190 Val Ala Asp Cys Phe Asn Ile Thr Val Thr GluTyr Ser Ile Gly Pro 195 200 205 Ala Ala Lys Lys Asn Thr Ser Glu Ala ValAla Ala Ala Asn Gln Thr 210 215 220 Glu Val Glu Met Glu Asn Lys Val ValThr Lys Val Ile Arg Glu Met 225 230 235 240 Cys Val Gln Gln Tyr Arg GluTyr Arg Leu Ala Ser Gly Ile Gln Leu 245 250 255 His Pro Ala Asp Thr TrpLeu Ala Val Leu Leu Leu Leu Leu Thr Thr 260 265 270 Leu Phe Ala Met His275 35 762 DNA Homo sapiens 35 atggcgaacc ttggctgctg gatgctggttctctttgtgg ccacatggag tgacctgggc 60 ctctgcaaga agcgcccgaa gcctggaggatggaacactg ggggcagccg atacccgggg 120 cagggcagcc ctggaggcaa ccgctacccacctcagggcg gtggtggctg ggggcagcct 180 catggtggtg gctgggggca gcctcatggtggtggctggg ggcagcccca tggtggtggc 240 tggggacagc ctcatggtgg tggctggggtcaaggaggtg gcacccacag tcagtggaac 300 aagccgagta agccaaaaac caacatgaagcacatggctg gtgctgcagc agctggggca 360 gtggtggggg gccttggcgg ctacatgctgggaagtgcca tgagcaggcc catcatacat 420 ttcggcagtg actatgagga ccgttactatcgtgaaaaca tgcaccgtta ccccaaccaa 480 gtgtactaca ggcccatgga tgagtacagcaaccagaaca actttgtgca cgactgcgtc 540 aatatcacaa tcaagcagca cacggtcaccacaaccacca agggggagaa cttcaccgag 600 accgacgtta agatgatgga gcgcgtggttgagcagatgt gtatcaccca gtacgagagg 660 gaatctcagg cctattacca gagaggatcgagcatggtcc tcttctcctc tccacctgtg 720 atcctcctga tctctttcct catcttcctgatagtgggat ga 762 36 31 DNA Artificial Sequence Description ofArtificial Sequence N-terminal sense oligo 36 tttggatccg atgcaaggaggtggcaccca c 31 37 29 DNA Artificial Sequence Description of ArtificialSequence C-terminal antisense oligo 37 caagaagctt tcagctcgat cctctctgg29

What is claimed is:
 1. A purified monomeric β-form of a prion proteinwhich has more β-form than α-helix structure and retains solubility inaqueous solution in the absence of a denaturant.
 2. A purified monomericβ-form of a prion protein which has more β-form than α-helix structureand retains solubility in aqueous solution in the absence of adenaturant and which is non-aggregated, but capable of forming anaggregated fibrous and/or amyloid form.
 3. A purified monomeric β-formof a prion protein which has more β-form than α-helix structure andretains solubility in aqueous solution in the absence of a denaturantand which is non-aggregated but is capable of forming an aggregatedfibrous and/or amyloid form on exposure to denaturant.
 4. A purifiedmonomeric β-form of a prion protein which has more β-form than α-helixstructure and retains solubility in aqueous solution in the absence of adenaturant wherein the β-form of the prior protein will interconvertbetween predominantly α-helical form of a prion protein and said β-formof a prion protein when in aqueous solution at a concentration of morethan 1 mg/ml and in the absence of a denaturant.
 5. A purified monomericβ-form of a prion protein which has more β-form than α-helix structureand retains solubility in aqueous solution in the absence of adenaturant wherein the β-form of the prion protein will interconvertbetween predominantly α-helical form of a prion protein and said β-formof the prion protein when in aqueous solution at a concentration of atleast 12 mg/ml and in the absence of a denaturant.